This all changed when I chose to pursue my PhD in Okinawa, an opportunity that brought me very close to the sea. Residing near the ocean has been an enlightening experience, offering me the freedom to engage in marine sports at my leisure. The seas around Okinawa, known for their stunningly pristine corals and a diverse marine life, lie just at the edge of the coral triangle, making them among Japan’s most exquisite.

Prior to my move, I prepared for a new hobby I had long anticipated by obtaining a scuba diving license while at home in Bali. This was a novel and exhilarating venture for me, especially since my family never shared an enthusiasm for such activities, leaving me somewhat isolated from these experiences.

Upon my arrival in Okinawa, I immediately immersed myself in the underwater world. I purchased diving gear within my first few months and quickly connected with fellow diving enthusiasts, some even more passionate than myself. This marked the beginning of my aquatic adventures. I seize every opportunity to dive, always adhering to the rule of never diving alone – it’s essential to have a buddy!

Balancing diving with my PhD has been challenging, especially with frequent business trips limiting my time at sea. Despite these constraints, I make it a point to dive as often as possible, typically around twice a week. Diving here is also quite affordable; renting a tank costs 800 yen (previously 600 yen, but inflation has its say), good for about 60 minutes dive time.

I have several favored diving spots, but I’m particularly fond of the sandy bottoms and channels around Seragaki. I feel like I’ve encountered nearly every type of marine life here, except for sharks, which remain elusive. I also enjoy photographing the underwater world, and below are some of the shots I’ve taken with my trusty RX100-M1 at Seragaki, including my favorite picture of a turtle from a dive where we spotted about five turtles.

Then, there is the clownfish that was quite shallow, this was also very nice.

Finally, I took a picture of this cuttlefish almost 30m deep. It was as if it was trying to get our attention for pictures!

And there are some of the best pictures I took in Seragaki. I hope I will be able to get even better pictures in the future!

I’ve heard really good reviews about EMBO workshops which compelled me to sign up. At first, I was reluctant because I feel like it was a bit redundant and less relevant for me, as I have had experiences with library preparation and bioinformatics analysis. Nevertheless, I figured that I might learn something different, especially about the experimental part. Also, I was more interested in epigenetic sequencing, which unfortunately was not included in the workshop except for one or two lectures. Nevertheless, I had an amazing time there and did not regret joining the workshop at all. Definitely will go again for another workshop if I ever find something more relatable!

EMBO Practical Course 'Single-cell omics: deeper to genomics' started yesterday 🥼🔬🤩 Here's a few snapshots from Day 2 ⬇️ Four more days to go!#EMBOSCOmics pic.twitter.com/xjyGQsk89F

— EMBL Events (@EMBLEvents) November 14, 2022

About the workshop

The content of the workshop

It was a week long workshop consisting of hands-on experimental and computational practicals, with some lectures between. The main techniques used during the workshop were:

• DNTR-seq: Direct nuclear tagmentation and RNA-seq (genome and RNA-seq on the same single-cell)
• Strand-seq: Sequencing of DNA template strands during DNA replication In which both are done in single-cells.

Further, we also had lectures that include single-cell epigenetic sequencing and single-cell sorting techniques among others. I think it’s definitely a good workshop for people who will be doing these experiments in their projects.

The people

By the people I mean the instructors and other participants. They were some of the greatest people I’ve ever met and we really got along with each other.

Most of the attendees were from Europe, which is probably because of proximity. I also noticed that all of the participants were wet biologists, with most having barely no background in computational work. It was nice that we were able to share our experiences dealing with different things, as we all have different backgrounds. The instructors were also really helpful along the course. We went out downtown for dinner and drinks which was super nice.

In general, everyone was very supportive and friendly, definitely made it worthwile attending the course.

During the workshop

While the workshop itself was comprehensive, I feel one of the most important part of the workshop was networking. We were able to talk to each other and also the instructor during the coffee breaks, lunch and dinner. I have to say that their food service was amazing.

Some comments

While the workshop was very nice and interesting, I feel that the schedule was too packed, and we didn’t have enough rest. For me, I wasn’t able to be fully functional because I was just too sleepy during some of the occassions.

The poster session was also not very stimulating, we had limited time to present our own posters and no time at all to discuss in front of the posters. That was unfortunate but at least we were able to talk during breaks and dinners.

Other than that, it was a very nice workshop, and I was able to connect with people which I feel was the most important part of the course.

About Heidelberg

Heidelberg was a very small city, I was a bit surprised that I probably went to most places in one day. It is famous for the Heidelberg castle which was really cool. Unfortunately, it was very foggy most of my time there, maybe I should visit again in the summer. The timing was also a bit early for Christmas market so I wasn’t able to enjoy that.

As I only had several free days, I only walked around Heidelberg, here were the places I visited:

• Downtown Heidelberg
• Schloss or the castle
• Philosophers’ walk
• Bars

It was totally different from Japan, and I think that it was a very beautiful city.

Downtown Heidelberg

It’s amazing! I’ve never been to other cities in Europe, but it totally feels like Europe.

Schloss Heidelberg

This is probably the top tourist spot in Heidelberg. I think it was a very relaxing and nice spot.

 

Philosophers’ walk

This is a hiking (walking?) path in the outskirts of the downtown, which is reachable by walking from downtown. Through this path, we can see the whole city, unfortunately on that day it was too foggy and I wasn’t able to see anything. But, it was a really nice walk even though it was a bit tiring to go up the stairs.

  </div> ### Bars! The night life at Heidelberg was really not bad. The bars were very lively and had nice beer for cheap (well not expensive but not super cheap either). After the workshop, we went to several bars to get drinks, unfortunately we were too tired and had to leave early.

### Beer beer and beer I've heard stories about German beer being really good so I did try it. It was amazing. Well, I've had similar stuff in Japan, but it's the norm there.

 

### The food The food was really great as well, but very very heavy, and everything tasted similar. I think I started wanting asian food after my 3rd day there hahaha. I particularly loved the schnitzel and the pork knuckle. Here are some of the places I thought were worth visiting: First, I was recommended by the tech in my lab to get schnitzel at this place. I ended up going here twice because I really liked it. The portion was generous, the waiter was really nice, and the food was awesome. I enjoyed the mushroom sauce Schnitzel. Weinstube Schnitzelbank: https://goo.gl/maps/1NujA1Fu7oSgncSW9 Here is the picture of the garlic sauce schnitzel:

Next, I wanted to get pork knuckle and to have some beers. Ended up going to this place, and I have to say that the pork knuckle was really great. Vetter's Alt Heidelberger Brauhaus: https://goo.gl/maps/RrfJHd9dCzyBUejv5

## Conclusions This was a very nice trip, my first time in Europe and definitely was totally different with what I was used to. I highly recommend the practical course, and I believe that other EMBO workshops are at the same level.

Just to summarize my paper, it’s a study about the transcription factor (TF) NF-kB. This transcription factor translocates to the nucleus upon cell activation, in this case B cell. I visualized the formation of molecular aggregates that occur in the nucleus that involve NF-kB, DNA and other transcription related proteins using fluorescence microscopy, and finally predicted that the generation of enhanced genomic interactions because of this aggregates eventually lead to variability in gene expression.

The long path to publication

While it may sound straightforward. Reaching the end of this project took quite a bit of time and a lot of trial and error. It took me 3 years, from being a clueless undergrad to being somewhat knowledgeable in the field. (I still think I don’t know a lot of things tho!).

How it all started

It all started when I was an undergrad, looking at labs for my 4th year thesis project. I was told by my former PI that there was a project that involved fluorescence imaging, in which I can be the sole investigator. Moreover, I also had around a month of free time before the start of my undergraduate research and being involved in this project meant that I can do some kind of work at that time. This project was very exciting to me as it gives me independence, which might be good or not depends on how you look at it, but for young me, it was an opportunity for me to give my best in completing the project.

I initially planned to leave Japan at the end of my undergrad, but at that stage, we realized that we did not have enough data to complete the paper. I then decided to stay for Master’s, initially only to finish the research project and publish a paper. I’m glad I was able to secure a scholarship from the Honjo International Scholarship Foundation, so that I didn’t need to worry too much about funding during my Master’s.

Initial ideas and plans

At first, the project was simple. To investigate what these nuclear aggregaets are, and how they affect gene expression of target genes. That’s it, and so I had to do an extensive literature search on the possible mechanisms. Here, I was taught about the concept of super-enhancers, where multiple enhancers in close proximity can work together through the aggregation of transcription factors and other proteins, to activate a higher level of gene expression.

The discovery and change of ideas

By reading more and more papers, I got several ideas on the biochemical properties of these aggregates. At first, I was just pretty much following orders, and analysed the data in a way that does not fully utilize the data. After a while, I was more comfortable with programming and felt more free in exploring the data, where I found that some genes showed different patterns of gene expression across doses. This finally became the main topic of discussion of my paper.

Things that did not go according to plan

“Broken” microscope

The microscope I used throughout this research was mostly the confocal microscope given from RIKEN. This was transported all the way from the outskirts of Tokyo to Osaka. After setting-up the microscope, I found out that the images produced were highly noisy. Later, they found out that the engineer installed a filter backwards, and I ended up wasting a month or so here waiting for the people from the microscope company to fix the problems.

After a couple of months, I realized there was another trouble where the laser got very weak and the images produced were highly noisy. This time, the fix came quite quick, and I was able to continue experiments straight away after 1-2 weeks. If I’m not mistaken, some of the mechanical parts were moving around and thus the efficiency was reduced.

Software dependencies

Reproducibility has been plagueing bioinformatics research since forever and my limited bioinformatics knowledge at that time was in no way immune to that. If you are concerned about this, you might want to check this paper out!

The problem at that time was because of COVID, we had to work from home and I tried to switch to a different high performance computing environment in the middle of my research. I tried to replicate my previous environment including software versions. Here, I tried to use conda, but it turned out to be a disaster as some software actually showed different computational results. While most of the differences were minor, they were large enough to change the outcome of the major genes I was focusing on.

At that time, I learnt everything mostly by myself and certainly no one ever taught me about reproducible research. This is one of the reasons why I chose to move here to OIST, where I’m learning to create reproducible bioinformatics pipelines. Currently, I try to containerize my work most of the time using Docker and Singularity, when working with R, I always try to use renv, and I always try to use nf-core to run NGS pipelines!

COVID-19

COVID-19 hindered my progress by quite a bit. Firstly, it was difficult to work from home, while I had a pretty decent PC back then, it was still difficult as it was not as good as the PC at the lab. I also had to access my data remotely, which was not really convenient. However, what was most difficult was the inability to do wet experiments. As at that time I still needed more data, it was quite a hindrance when we were unable to go to the lab for several months.

Creating a mutant cell line

With the cell line I was using, immunofluorescence (IF) was almost impossible. At that time, I was trying to figure out the co-localization between NF-kB (GFP) and BRD4. At first, we thought we did IF properly and we were able to get decent amount of co-localization between these proteins, but I realized that most of the signal was just autofluorescence of the GFP and thus were artifacts.

Well, that was not good, so I discussed with Prof. Akira Imamoto from Chicago University to ask about creating my own cell line that expresses RFP-tagged BRD4 proteins. I found out that it was not that simple, as it included sooo many steps to confirm that the experiments were not flawed or biased. I firstly needed to design the experiments using cloning software, sequence all the vectors I obtained from cloning, and of course sequence the cell lines overexpressing the target protein upon transfection.

I used the Gateway cloning technique to incorporate the genes expressing for the BRD4 proteins into an expression vector. I actually had some problems along the way as I was using a fairly old reagent and found out that some of the clonings failed multiple times because the efficiency was too low. I’m glad that I was able to solve this problem after using unopened (albeit old) reagents which had fairly higher efficiency.

I also had problems with transfection efficiency, where it was less than 50% for DT40. Despite that, I managed to get a single clone after using serial dilution to culture transfected cell on a 96-well plate, it took me more than 3 months in total to finish these experiments.

Failed CRISPR-Cas9 experiments

As I was predicting DNA-DNA interactions, I wanted to validate if these regions actually behave like how I predicted in-silico. I resorted to using CRISPR-Cas9 to induce long-deletions in the genome, spanning putative enhancer regions. I was lucky that my lab had connections with Prof. Kawaoka at Kyoto University (currently in Tohoku Univ.). I discussed with him about the strategies needed to remove these putative interacting genomic regions before performing further gene expression validation experiments.

With the experience I had from making overexpression cell lines, I was up for the challenge. I was so wrong to think that this time, it was gonna be easier, it never got easier. Firstly, to remove a certain stretch of genomic region, I would need to target 2 different sites of deletion, where the Cas9 proteins can induce double strand break and by chance non-homologous end joining may occur. As I was targeting multiple targets, and that for each target I prepared multiple guide RNAs (gRNA), I ended up with so many combinations of plasmids which I needed to make. This is also caused by the difficulty in transfecting the cell line, which required me to design plasmids that each include 2 different gRNAs, as transfecting 2 different plasmids with single gRNA is too inefficient and is probably statistically almost impossible.

Indeed, while the cloning experiments were relatively optimized and rather easy, it was still very very laborious. Further, I had to get single-cell clones which took weeks, and sometimes the clone only have half of the chromosomes deleted, in which I needed to do another round of selection.

These experiments took me in total almost half a year to complete, only to realize that somehow the cells are not doing well after the deletion of some minor genomic regions. I wasn’t even able to induce deletions at some of the regions I targeted. So, the CRISPR-Cas9 experiments were pretty much a failure, but I learned a lot and implemented the techniques to help other people too!

Review, review and review

We decided to submit the paper to (Review Commons)[https://www.reviewcommons.org]. Review Commons is a platform where revisions from the first round of review can be transferred to any one of their affiliate journals. In general, it is supposed to reduce review time, as manuscripts can be transferred with a click of a button to journals after the first round of revision.

First revision

After around 2 months, we received the first set of reviews from a total of 2 reviewers. The first reviewer was fairly supportive of our results and only demanded a bit more changes to strenghten our discussions. Reviewer 2 however, was more critical of the results and demanded quite a bit of further exploration on the data and some additional experiments to prove our points.

In general, we were told that the conclusions and results taken over the course of the paper was not conclusive and did not show major “conceptual advance”. At this stage, I do agree with most of the comments given by the reviewers. Therefore, we decided to do a major revision, where we add new data and perform many new experiments. These additions added quite a lot to the paper, it went from (this)[https://www.biorxiv.org/content/10.1101/2021.07.13.452147v1] to (this)[https://www.biorxiv.org/content/10.1101/2021.07.13.452147v2].

Submission to PLoS Genetics

As the paper was on Reviewcommons, we were able to submit to a select number of journals. We firstly submitted the paper to another journal, which is actually one of my favorite journals. Unfortunately, it was rejected, as it lacked “conceptual advance”. Maybe it was, or maybe the editor were mostly concerned about the first round of reviews, and did not consider the additional data and results we brought forward.

Then, we submitted the paper to PLoS Genetics, which was also a very good journal, but I felt like it was a bit out of topic, as my paper wasn’t really about genetics. Nevertheless, it went through, and after 2-3 weeks it was under another set of reviews. The 2nd round of reviews were actually very helpful, as the reviewers pointed out some of the weaknesses in our paper which we agreed. Thus, we added further justifications and analysis, and alter some of the text. However, weren’t really delighted about a 3rd round of review, which did not add anything useful to the paper and took us another 1.5 months.

In total, after about a year of the review process, our paper was finally formally accepted and is now published. Honestly, it could’ve been faster, but I guess it’s not too bad for my first publication.

Explaining the paper

I will now explain the paper step-by-step, to make it (hopefully) easier for people which are not as well-versed in this field to understand.

Introduction

Basically, this research builds up on the previous papers by Shinohara et al., and Michida et al. I feel that the latter paper could have explored the data more, as they tried to use single-cell data as bulk data, averaging the expression data and does not capture single-cell expression dynamics.

Therefore, in my research, I aimed to utilize the single-cell data as much as possible, while adding experimental data to validate the results. By investigating the data more intensively, and leveraging the single-cell resolution of the acquired data, I was able to obtain interesting new insights about heterogeneous gene expression. Previously, the analysis was averaged across all of the cells, losing single-cell resolution which is highly important in immune cells such as the B cell.

Materials and Methods

As explained before, I performed all sorts of experiments along the course of this research. This includes everything from computational sequencing analysis, to molecular cloning.

Firstly, fluorescence imaging experiments and analyses. Here, I took over the 2nd author’s work, Inaba-san at RIKEN, where they were analyzing the dynamics of the formation of the NF-kB nuclear aggregates dynamics. As the basic protocols were pretty much established, I had not much problem. For the analysis of the imaging data, I was also handed over an ImageJ macro to quantify the foci. While there was no problem with the codes, I had to add some modifications to make it easier to understand, as well as creating automated visualization methods using my language of choice: R. This quantification and visualization method is currently in press in the Methods in Molecular Biology book series.

Secondly, single-cell RNA-seq. I was also lucky that the data for this part was already taken several years ago and thus I didn’t have to take it by myself. Here, I mostly used Seurat, to determine the cellular states of single-cells. Of course, being my first time handling any sort of sequencing data, it was a bit difficult as I didn’t have the basic knowledge on sequencing analysis. However, I ended up learning a lot about sequencing analysis, especially fine-tuning parameters to extract the most important information out of single-cell RNA-seq data, including pseudo-time/trajectory analysis of NF-kB activation in B cells in this case.

Thirdly, single-cell ATAC-seq. Honestly, I also went in blind when we took the scATAC-seq data. At that point in time, I didn’t really understand what we could accomplish using single-cell data as opposed to bulk data, especially that we only took 2 different dose conditions; both of which shows pretty much homogeneity within samples. Took me a while reading papers until I found a paper by Pliner et al where the author showed that scATAC-seq data can be used to predict cis-regulatory DNA interactions. From here, I expanded my analysis to try the find the relationship between DNA interactions and the patterns I saw in the scRNA-seq dataset.

Other than that, as explained above, I also performed molecular cloning to create mutant cells that express genes I needed using the PiggyBAC system, and also CRISPR-Cas9 experiments using the high-specificity enhanced specificity Cas9. These experiments took most of my time, especially because the nature of the cells I’m working with.

Results and conclusions

To sum up the results and conclusions, we were able to characterize the NF-kB foci, where they show condensate-like properties. We also found that the interactions between NF-kB and BRD4 may be crucial in governing transcriptional dynamics in super-enhancers.

Future prospects

In the future, I was expecting that someone will continue this research to inclue non-coding RNAs and their function in regulating NF-kB condensates. The data is there, I laid some of the groundwork to analyzing the data, but unfortunately I’m not sure anyone in my previous lab was interested in continuing this line of research. Furthermore, I also think that the interaction between BRD4 and NF-kB is unique and that some research could be done in understanding that interaction, especially where BRD4 inhibition causes the upregulation of NFKBIA, the negative regulator of NF-kB. I also think the incorporation of genomic interactions such as Hi-C would be able to resolve some of the questions that remain unsolved.

I have finally finished my Masters in Osaka University and is currently. There were quite a lot of hurdles during my master’s study, mostly with research, and the pandemic. Nevertheless, it was largely fruitful, I was able to finish with 4 publications as the first author: a research paper, which is currently under minor revision, a review paper which is also under minor revision, a book chapter about fluorescence imaging methods which should be published soon, and another book chapter in Japanese about single cell analysis. Now, I’m on my way to Okinawa, Japan, to start my PhD at Okinawa Institute of Science and Technology (OIST), which is a new and very beautiful campus surrounded by spectacular beaches.

In this post, I will be mostly writing about the process on how I was able to secure a PhD position. I pretty much got accepted to all the PhD programs I applied to (well, honestly, I only applied to 2 places) and I almost got an offer for a job (at least I think so). Hopefully, my experience will also be helpful to those still thinking about going for PhD too!

Why bother going PhD

Embarking on a PhD is not an option for most people. It will easily take 3-5 years of hard labor and low pay; some might even break your bank! It’s just probably not an option if you want to settle down and earn money. That’s fine, I think, a PhD might not help much if you want to go to the industry. But for me, I want to learn more, contribute to science, and to do science independently. Furthermore, I couldn’t care less if I were rich or not, that may come later. These reasons led me to where I am now, doing my PhD.

Applying to a pharmaceutical company

At first, I was thinking about getting a bit of experience by working in the industry for several years before embarking on a PhD, at this point my research was kind of stagnant and I felt like it could also serve as a good break. So, at the same time with my PhD applications I decided to apply to a pharmaceutical company which did all screenings in English as I couldn’t be bothered to do those stuff in Japanese. Anyhow, at this point, job hunting was pretty much over, and I had not a lot of options left.

I then prepared my CV and motivation letter without double-checking and submitted them. Luckily, I was invited for the first interview, probably because they were looking to start a new department which coincides with my field of expertise: computational biology. Then I was invited to a first interview, where I talked about my research. Here, I felt that they were really interested in hiring me. Though one thing really made me doubt, when I asked about the computational infrastructure available, they did not have any yet and expected me to initiate it. This was beyond my expertise and felt like I can’t develop myself much here.

After the first interview, I waited for several weeks and got an invitation for the 2nd interview. By this time, I ended up getting some nice results and was close to getting my research published (or at least close to submitting a research paper). At this time, I thought that I might as well just go straight to PhD. I also has a bit of thought that being in a Japanese company means that I will most likely be exposed to a similar environment, which eventually set me back.

After thinking for a while and discussing with family and friends, I decided to withdraw my application and focus on getting a PhD position, as I believe that I still have so much to learn. Well, the industry can always be an option in the future in case I fail academia.

Considerations for PhD programs

For my PhD, I was thinking about somewhere I can do cutting-edge basic research. I’m not really interested in applied research. Of course, I also took the environment (be it research or social) and the benefits, while most importantly the research topic and the PI into consideration.

At first, I had a short list of PhD programs I was planning to apply for in order of preference:

• European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
• Center for Genomic Regulation (CRG), Barcelona, Spain
• École Polytechnique Federal Lausanne (EPFL), and
• Okinawa Institute of Science and Technology (OIST)

I have also contacted some of the PIs at some of these institutions and some of them responded nicely. However, I ended up only applying for two, which is primarily because of getting accepted to OIST early on. OIST checked most of the boxes that I looked for in a PhD program. At first, OIST was my backup as I really wanted to leave Japan, being here for a total of 6 years at the time of my graduation. However, things changed dramatically along the way, and I believe that I made the right choice.

PhD applications

Okinawa Institute of Science and Technology

I applied for OIST early, as it is a 5-year program and requires only a bachelor’s degree, meaning I could apply during the first year of my masters. I remember applying for the September 2021 intake and requested to defer. At first, I did not think much about it as it was just a backup. I went there a couple of years ago and the campus was fantastic, but it wasn’t really a first choice as I wanted to leaving Japan.

Fortunately, I was invited for the admissions workshop, including interviews with some of the faculty members. We were supposed to visit the campus, but unfortunately it was still COVID, and they cancelled the on-campus admissions workshop. We had to attend presentations about student support online, even the virtual lab tour got cancelled due to the current situation at that time. The financial and student support in general is probably one of the most generous in the world and got me a bit interested. Then, during the interviews, I remember getting asked about my motivation in coming to OIST, and about my research. I had some very meaningful scientific discussion with some of the PIs and that sounded great. Nevertheless, what made me sure that I want to go here was after talking to my current rotation lab PI, Nick Luscombe. After talking to Nick about. He also seemed very enthusiastic in taking me in as a PhD student. He seems to also be a very good mentor and is a well-known scientist in the field of computational biology.

Then, about the research project, I had doubts about working with planktons, you can’t really see them, and they don’t seem significant. However, I learnt later that they possess interesting genomic structures. The “same” species have scrambled around genomes, and that the mechanism is not yet clearly understood. This is quite cool as it can possibly lead to discoveries of a new mechanism of gene regulation, which might or might not be applicable to humans.

Moreover, I am also expected to learn about the basics of reproducible bioinformatics research, which I never had back in Osaka. Most places expect results without process, and this is not that place, which I think is ideal for a self-taught bioinformatician like me.

For me, I have always wanted to study something that is “useless”, e.g., dinosaurs, but I never had the chance to. While not necessarily as “cool” as dinosaurs, I think that this is a good step for me to take as I will have the chance to work with non-model organisms, and to learn the basics of bioinformatics that I can apply to more biomedical projects in case I’m going back to biomedical-oriented research.

Aside from that, it is interesting to have lab rotation on the first year. Here, I get to try different research, even something that’s out-of-field. This means I can learn and experience new things and decide what’s best for me. This was also quite pivotal for me in my final decision.

One major drawback though is that this university is in the middle of nowhere. It is surrounded by beautiful beaches and the air is very clean, sure, but without a car it is tremendously difficult to live a normal life. Currently, I still don’t have a car and is trying to survive with a 50cc scooter.

École Polytechnique Federal Lausanne

École Polytechnique Fédérale de Lausanne (EPFL) was also on my list. This institute is a highly established institute which has a good reputation. They have really good computational infrastructure and also connection with the University of Lausanne. The salary is also quite high, while the cost of living is quite high as well. I ended up applying here because the application was also open quite early like OIST. As usual, I prepared my CV and my recommendation letters from my current and previous professors and submitted them to their online system.

Then, after a while, I was invited for an interview, where I presented about my work on transcriptional regulation in Osaka. I was also able to talk to PIs that might be interested in recruiting me as a PhD student. However, I saw that their research was highly applied and biomedical, and I wasn’t really interested in that. I felt like I could use a break doing something else different.

After the interviews, I was told that I was accepted to the school, but I still had to find a PI in a year, as I couldn’t find a suitable PI during the admission process. The next month, I was contacted by one of the PIs if I would like to work with them on a project, which while to an extent, relevant to my previous project, I wasn’t really interested.

Therefore, I gave up on EPFL and decided on OIST. Which might have been the right choice.

What now?

Fast forward to 2022, I arrived at OIST. It’s a small community, everyone knows everyone, and nobody feels distant, everyone is so welcoming! I got new friends, and my first few weeks have been great. The view is spectacular, the beaches, the labs, the buildings, everything is just perfect.

What ultimately helped me land a PhD position, some tips from me

Indeed, applying for PhD is not that simple, I’ve heard people who applied to more than 20 places and only got 1-2 offers. So here are some of my advice, they may be highly opinionated, but got me through my PhD application processes.

First, understand your research inside out. You should be able to explain what you did during your research and how important is it in your field. Not only that, but also how it can be developed further and the potential implications. Then, do not talk beyond your capabilities and be eager to learn new things, basically don’t lie, most people applying are probably on the same level as you. Finally, you should be curious. Ask questions. Asking questions will spark further discussions between you and whoever it is interviewing you. On top of that, you should have a clear goal on what you want to do in the future, be it in the academia or in the industry. Good luck!

Learning new experimental techniques

Well, things started to get boring in my research project and I challenged myself to do cloning experiments. For those of you who do not know what cloning is, basically it involves creating an expression vector (which in my case is a plasmid) which can be introduced to a host cell and induce wanted gene expression. For example, you can introduce an expression vector containing GFP (Green Fluorescent Protein) to a cell and they will glow under GFP excitation spectrum. I think some of the DNA vaccines currently used for COVID-19 follows a similar principle so you can google that out!

Unfortunately, nobody in my lab is currently doing cloning and I was pretty much left to figure things out by myself. On the other hand, I had a supervisor from another university with a research collaboration which helped me a lot during the experiments, even though only through email and Zoom. Nevertheless, it was a really fruitful experience. I had zero experience when I started causing me to end up failing a lot of experiments, but it was very rewarding as now I got a new skill and I can now plan cloning experiments by myself.

After that, using the same technique I also started doing CRISPR-Cas9 experiments. These experiments turned out to be very simple, you just need to get the information on the genomic position you want to knock out, design your guide RNA and proceed with cloning to a suitable vector. It’s very simple, no wonder it changed the field of genome editing and won some of the pioneers in this technology a Nobel Prize.

If you are interested, maybe take a look at this page: it contains most of the information you will need in designing your own CRISPR-Cas9 experiments!

Addgene: Zhang Lab CRISPR Page

Getting my first conference award

As I said before, I joined an academic conference which was unfortunately held online. It was a domestic conference on Biophysics （生物物理学会）. It was supposed to be held in Gunma, but unfortunately as we all know due to COVID-19 restrictions, it was held online.

My impression on the conference was that it was not really fulfilling, I had 0 interactions with other people which was really bad considering academic conferences are usually where you get to know people in your field. The organizer lets us upload a poster where people can comment and ask questions. However, it was designed in a way that makes asking questions sort of difficult, it’s kinda hard to explain, but I would say it’s more like commenting on a Facebook post and not like a Discord chatroom (not sure if this is a good example tho LOL).

As we all know, Japanese people also don’t tend to ask a lot of questions, especially when dealing with foreigner, it might also be why I basically had 0 question on my poster hahahaha. I wrote that it’s fine to ask in Japanese tho… Guess there’s also an impact of having the conference in that kind of format. Maybe having private Zoom video chat rooms will be more interactive?

I guess nobody was to blame, it was their first time doing the whole thing online. On the other hand, I do think that they need to change the format if they want to do another online session in the future.

Well I got a poster presentation award after getting some senior researchers look. I don’t know if this is good or not but at least I got something from this conference…

How I managed to write a journal article in Japanese

A couple while ago I was invited to write an article about single-cell epigenomic analysis by my professor and I was like ok I’ll do it.. except that it was supposed to be in Japanese and I have never actually wrote an actual writing in Japanese.

Let me briefly explain the contents of this article. I mostly discuss single-cell RNA-seq and ATAC-seq techniques and its applications on understanding epigenetic regulation in immune cells. I also write about currently available analytical techniques and how it is used to uncover complex immune cell differentiation through an epigenetic perspective. I took the time to try and make the article easy for most biologists, even those coming from different backgrounds, so hopefully it will be easily understandable for readers.

I had quite a hard time writing this article actually. Firstly, I wrote things in English, then I had one of the assistant professors help me with translating my writing to Japanese. Finally, we discussed around the translated version. At first it was a bit difficult, but revising the work was not really as hard as making it, somehow 1.5 years of Japanese working environment was enough for me to adequately understand technical terms and Japanese writing style. I’m sure that this won’t apply only to me, I firmly believe at the saying “when there is a will there is a way” and I think this proved that.

So here’s the resulting work, it is published in the Journal of Clinical and Experimental Medicine (医学のあゆみ) under the title “prediction of transcriptional regulation in immune cells through single-cell analysis (免疫細胞の１細胞解析による転写制御機構の予測)”. Unfortunately it is unavailable online, but let me know and I might be able to hand you a copy!

医学のあゆみ　276巻10号　2021年3月6日

I am also thinking about writing a similar article on my blog in English as I am quite sure most of the readers here do not speak nor read Japanese, let me know if anyone’s interested!

Getting back up after failures

As I said before, I have had my share of failures in the last couple of months. Nevertheless, it wasn’t necessarily a bad thing as I learned quite a lot. It used to take me more than 2 weeks to finish a cloning experiment but then now I can come up with nice results within a week. I guess the moral of the story is to never give up. You just don’t know what lies ahead.

So what’s up next?

Right now, I am currently preparing for my first peer-reviewed publications. It took me quite a while to be able to get enough results for one but I guess I am quite relieved that I was finally able to make decent progress.

I am also currently looking for PhD positions abroad or maybe in Japan. I’m more interested in a position abroad tho, as I’d like to get some experience in a new environment. Japan had been great, but I think it’s time for me to get a broader perspective on how the research field works. Just hoping that I will be able to learn even more in my next post. I’m also open to working in the industry, as long as I can learn even more things.

See you next time!

Before, I might have thought about leaving this university to seek for new experience, nevertheless I found quite a strong reason to stay for another 2 years. This reason is basically as I have only performed research for a year, I haven’t gathered enough data to publish a research paper, and thus by staying for another 2 years, hopefully I can finish my research and publish a paper ASAP.

Staying for another 2 years, however, isn’t as simple as I could not extend my undergrad scholarship which means I need to find another source of funding. Just so you know, tuition fee here is 267,900 yen per semester and there is also a matriculation fee of 282,000, plus it costs around 100,000 yen per month for lodging and food. Currently, I don’t have that much money and thus I had only 2 options: to look for another source of funding or end up doing part-time jobs which will definitely sacrifice my research hours. Luckily, my professor was kind enough to let me do part-time job in the lab, basically performing analyses unrelated with my work to get a little bit more cash. Even then, this will not be enough to cover everything and thus I knew I needed to look for scholarships.

Actually, as I am graduating early, I also had another option, to stay another semester and graduating in September. This means that I can live off the current scholarship until I apply for another scholarship later on. I heard that there are more scholarships for international students around September as most international students follow the Fall entrance. However, I did not want to extend my study and I intended to attend the graduation ceremony at Osaka-Jo Hall (which was unfortunately cancelled due to the coronavirus outbreak).

So here goes my adventure:

Sato Yo International Scholarship Foundation

Firstly, I was offered to apply to the Sato International Scholarship Foundation scholarship. Apparently, this is the first year that the international office for G30 students received slots to recommend students for this scholarship and I was lucky to be one of the students getting recommended. This scholarship rewards scholars with 180,000 yen monthly stipend and 100,000 yen per year on travel cost if you join conferences. This should be enough to cover my living cost and to pay tuition fee.

The documents needed were basic documents such as the passing certificate, recommendation letter and forms, other than that we had to submit a typed research proposal and a written essay. So this was my first time writing essays and research proposal in Japanese so I had to ask around to help with Japanese. Anyhow, I was able to send the documents on time and I just had to wait for the results.

Entrance to Graduate School of Osaka University

As I had to submit the certificate of passing (合格証明書) for the Sato Yo scholarship around late September, I could not wait for the English course (SISC) which did not start the intake until later in December (which I was planning to apply before the scholarship offer). I was forced to take the normal Japanese exam (院試), and I was not prepared. If I had TOEIC/TOEFL, I could have applied for the 特別選 or the selection by recommendation which skips the written exam straight to interview, however the bad news was I had not taken TOEFL since high school. Plus, if I were to take the TOEFL/IELTS/TOEIC, I would not have had the results by the time of the application. So I was left with no choice but to take the normal exam. At least I was allowed to answer half of the exam in English (I still had to answer the English exam in Japanese).

The application was straightforward, I just had to go through a pile of documents in Japanese. For most part, I just had to fill out personal information forms and write about 2 pages of essay on basically what I plan to do in the near future and what I did during my undergraduate study.

The exam

I barely studied for the exam. I studied a bit by reading some parts of the Essential Cell Biology book around 2 weeks before the exam. The week before the exam, I had a fluorescence imaging workshop (link in Japanese) at Nishi-Akashi (Hyogo). I thought the workshop was going to be relaxed but boy I was wrong! I barely had sleep for the whole week and we had to work on imaging analysis for that whole week. I also forgot to bring the notes I made during my short study before going to the workshop so I ended not studying at all at the workshop.

The next day was exam and I just did not care anymore as I was literally exhausted. The written exam consisted of two parts: Subject (Biology and optional Math/Chemistry/Physics) and English test. Honestly, it was quite easy, given that you understand the Japanese, fortunately I understood 80% of the questions and was able to answer. The interview, however did not go quite as planned, I was asked about my research and somehow I could not answer some of the questions. At that time I was a bit prepared to either fail or end up bottom of the result list.

The result

The result was out several weeks after the exam and I was very surprised to find my name in the top 10 of the results list. Somehow I managed to get quite a high score in the English exam (I pretty much wrote everything in hiragana and katakana). Nevertheless, the only thing that matters is that I passed the exam and I can apply for scholarships!

The aftermath and application for other scholarships

Several weeks after the submission of the documents, I was notified that the results were on the foundation’s website. I looked for my ID but could not find it, tried to look again for around 3 times until I had to admit defeat (LOL), I did not pass the document screening and thus was not invited for interview.

After getting turned down, I went to look for other scholarships that didn’t need university recommendation to apply. I looked at Jasso’s scholarship list page and found out there are several scholarships that I could apply with my current condition (without university recommendation). Here are the scholarships that I decided to apply to:

• Nitori International Scholarship Foundation (80,000 yen/month) [100/?]
• Honjo International Scholarship Foundation (200,000 yen/month) [12/191]
• ITO Foundation for International Education Exchange (180,000 yen/month) [14/810] Square bracket indicates the [accepted/applicants] for previous years. For these scholarships I am going to write the steps below: In general, it is probably advantageous to have a JLPT N1 before applying to these scholarships.

Nitori International Scholarship foundation

We had to apply through web, enter personal information and simple questionnaires. After a while, they will contact if I pass the first screening and I had to take a web test, this test is basically an SPI test (Aptitude test), and I kind of had to ask people in my lab to help as I could not really understand the Japanese. However, I managed to clear the test (my scores were not that good tho) and was asked to submit documents. These documents include enrollment certificate, transcript, one page of essay (hand-written), Zairyu card+passport copy, letter of recommendation, copy of apartment contract and JLPT N1 results copy (optional). After submissions, I was notified that I had passed the document screening and will be interviewed in January.

The interview was held in the Nitori headquarters and the atmosphere was pretty calming, I actually made some friends and had quite a good conversation with the other interviewees, one of which actually had mutual friends in Tohoku University. The interview itself was only for about 10-15 minutes with general questions regarding my study, organizational activity, and future plan. I think it went smoothly and I was quite confident of the results.

Honjo International Scholarship Foundation

Honestly, this one was the least troublesome to apply and the online system was also quite good. Furthermore, not a lot of documents were required and none hand-written. In general, the documents required were similar with the Nitori scholarship. The length of research proposal was also restricted and I could only write some parts of my research in the research proposal.

The interview was held in the Ito-en headquarters in Tokyo, however there was no travel cost reimbursement, but we could opt for a Skype interview. I chose to have the interview at their headquarters, hoping to make a better impression. Arriving at the Ito-en headquarters, I was greeted by one of the staff which escorted me and another guy to the waiting room at the top floor of the headquarters.

The waiting room was huge with really nice sofa. However that made me really nervous as not a lot of people were there, only the interviewees and the staff from the scholarship foundation. We were given a can of green tea while we wait which was nice. On the other side of the room, there was a display showing all of the products of Ito-en including Tully’s Coffee. After around 15 minutes of waiting, I was told to enter the other room to be interviewed.

Contrary to the interview of Nitori, the atmosphere was quite tense as I was directly interviewed by the board at directors, the interview went quite smoothly for around 10 minutes. I was asked to introduce myself and was asked several questions regarding my future and my research. I was a bit surprised when they told me that my Japanese was good (I was expecting other interviewees to be twice as good).

 

ITO Foundation for International Education Exchange scholarship

This scholarship was not that competitive, and probably for a reason. There were a lot of Japanese essays to be submitted. Most of which had to be hand-written. Took me several days just to finish the essays and that is probably the reason. Most of the communications were done by mail and by phone but I think everything went pretty smoothly.

After about a month, I was notified that I was subject to another round of screening (interview) which was held in Tokyo and all the guide was by mail and I had to notify them by phone.

(Bonus) MEXT Super Global University scholarship

Halfway through the application, I was notified about the SGU MEXT scholarship which is a MEXT scholarship recommended by the university. This scholarship is basically a MEXT scholarship which awards 145,000 yen per month (If I’m not mistaken) and the tuition fee is fully covered. There is a caveat however, in which the scholarship is only valid for a year and the .

The procedure was quite simple, I just had to fill in some forms and have my professor make a recommendation letter. The documents were then sent to the faculty and then the headquarters office where the final selection took place.

No interview took place and it was solely dependent on the university’s selection committee.

The result

I later found out that I was not selected for this scholarship by a close margin, and thus I was quite disappointed at the results. However, at that time I was still hoping to receive the other scholarships.

Literally the day after the Honjo scholarship interview, I received an email notifying that I have been selected as a recipient for this scholarship. I was so happy and a bit surprised as this was the most competitive scholarship I applied to. Right after the notification, I replied that I will receive the scholarship and thus I needed to cancel the others.

At this stage, I was still waiting for the result of the Nitori scholarship. I later found out that I was also selected for the Nitori scholarship, however had to withdraw as both of the scholarships did not allow scholars to receive another scholarship.

I also cancelled the interview for the Ito Foundation for International Education Exchange scholarship the day after the acceptance of the other scholarship, so unfortunately I can’t give insights on the interview.

Thinking about it, I was very lucky that I failed the SGU MEXT scholarship or I wouldn’t have been able to accept the Honjo International Foundation Scholarship.

The support given by the Honjo International Scholarship Foundation has been fantastic. The orientation and welcome party were actually scheduled to be at the end of March, but had to be cancelled due to the coronavirus. Scholars also get invitation to attend networking events and other events hosted by the foundation.

So here are my tips for you who are applying for scholarships:

• Try to make your research understandable to people from out of your field by avoiding jargons and technical terms
• Be confident
• Have a clear vision of what you want to do and/or be in the future
• Never give up

Til then!