Pesach (Passover) officially begins tomorrow at sunset. Today, those who observe must clean their houses in case they have forbidden products (most grain-based foods are not allowed to be consumed)—so in Jerusalem, the holiday effectively starts today. For me, I have no plans, but my family is coming to visit (it’s the Easter holidays back in the UK). I may take them to Bethlehem or the Dead Sea or whatever is simplest. It’s an interesting feeling—no longer being such a stranger to this land. Six months being here has at least given me a few throwaway phrases in Hebrew and the basic navigation skills necessary for the confusion that is the Israeli public transport system.
Exciting developments in the lab this week. After slogging through many a statistics blog and function documentation, I have made it all the way through a (preliminary) RNA-seq differential expression analysis. The final output being the following graph:
The red dots represent genes that are up-regulated or down-regulated in my treatments compared to controls to a significance of P < 0.1. Over 50 hours of work for one measly plot. It’s a profoundly satisfying feeling when programs and functions start to work. When things work as expected it becomes all the more easier to tinker around with inputs and outputs and parameters in the pursuit of understanding the processes at each step of an analysis. I’ve also exported the list of the genes from this plot into an excel file. I will soon begin the exciting and hopefully rewarding task of searching for biological significance. The main open questions of my project have to do with the key players in pluripotency and whether they are affected by my gene/protein of interest.
Today I performed a Western blot for the first time. A time-consuming and delicate process I’ve been successful at avoiding for my entire biological career up until now. However, this week I had to bite the bullet and asked got my lab-mate to help me through this trying time. I have protein extracts from putative CRISPR-KOs clones that I needed to validate. The result is promising. Of the 6 samples I ran, one of them seems to have lost expression of my protein. I’ll need to run a loading control next week to make sure I actually loaded that sample’s protein, but it’s likely I’ll be pleased at the result. Regardless, I’ll keep culturing the clones from this CRISPR trial. If all goes to plan, by next week I’ll finally have the materials I need to complete the final experiments for a first publication.
The past week has been a challenging one. Thankfully I got the opportunity to spend the weekend in Eilat, the southernmost city in Israel. Drove down from Israel with some friends. The route took us along the Dead Sea and was an enjoyable journey—but perhaps this was because I wasn’t the one driving.
The city of Eilat itself is nothing special, but the Red Sea and its associated marine-life/views are spectacular. From the coastline you can see the mountain ranges of both Egypt and Jordan (and Saudi Arabia if you squint). I’ll definitely make the trip across the border at some point—to Sinai on the Egyptian side and Aqaba in Jordan. Went diving for the first time as well (below is me, chaperoned by a very intimidating Russian man).
My RNA-seq data is back. I’m learning how to process the data from scratch. Somehow, it’s simultaneously more difficult and simpler than I expected, if that is even possible. What I mean by this is that after the initial hurdle of becoming familiar with BASH and linux and installing/running programs from github, I can see very clearly what I need to do in terms of following the pipelines and workflows that comprise the seemingly countless RNA-seq tutorials. However, I did not foresee the amount of trouble-shooting and data-fitting necessary for each step of this lengthy process. I’m currently at the stage of running differential expression algorithms on my mapped and aligned reads.
My CRISPR-KO clones have been expanded and I am beginning validation. I need to send the genomic PCR products I’ve generated for sequencing to verify if I have successfully knocked out my gene of interest. It’s tough working with so many cell lines at once, and this is a big reason the past week has been so taxing.
Just arrived home from a conference in Munich on chromatin and epigenetic inheritance. Didn’t present anything this time round, but I had the privilege of attending some very interesting and thought-provoking talks. Of note, some great presentations of new single-cell analysis pipelines and methodologies. In particular, I’d like to highlight work by Fabien Theis at the HZM (Munich) where his team developed machine learning algorithms capable of predicting differentiation states in a temporal manner, just from an individual single-cell analysis snapshot. There were also many talks about 3D chromatin configuration and LADs/TADs—which are turning out to not be as game-changing as the hype would suggest. Epigenetics is in a ‘wild-west’ kind of stage. There are countless techniques and methodologies being developed to probe all the new forms of regulation that are being discovered. We are also learning there there isn’t really a deterministic ‘histone-code’, and that all of these things we’re learning about under the umbrella of ‘epigenetics’ are intertwined with one another and have a complexity we are only beginning to scratch the surface of.
That’s mainly what I took away from the talks. But other than a dreary proclamation of our general ignorance in the field, I am glad that there are so many open questions for talented scientists to sink their teeth into. What would we be without a challenge?
Nothing much with regards to lab work as I was away in Germany. However, I will thaw my CRISPR-KOs and grow them up for characterisation. I have a week and a bit left until Pesach (Passover), which is when I hope to be done with the bulk of cell line generation and validation. I’ll need to think about how and what experiments need to be done as soon as time permits.
I’ve had a bit of a tiring week. Now that I’ve started taking a class alongside my research, time has become all the more scarce. It’s difficult to know exactly when you’re making progress of how you have changed for that matter. I’ve been wondering that myself lately. A friend I haven’t seen in more than half a year came to visit me in Jerusalem yesterday and I think the way we interacted was a helpful comparative proxy for the type of person I was and the type of person I have become. It’s only when you hold some variables constant can you with greater confidence observe what is different.
Domestic life and duties are getting to be routine. I know where to get food and supplies, how to organise my time that I cook enough meals and keep my apartment reasonably hygienic. My only complaint as of now with my day-to-day life is the commute I take to work each morning. As am still living in university dorms in another campus, I must travel across Jerusalem everyday to get to my lab. Public transport is not ideal in Israel at the best of times, and it can take from 40 mins to an hour in rush hour traffic. That’s two hours of my life everyday stuck on a bus. It doesn’t help that drivers here seem to not acknowledge the existence of break pedals. It’s bumpy and loud and the seats aren’t comfortable. I’ve tried to find ways to make my daily commute more pleasurable, music, phone games etc., but it doesn’t change the fact that it’s still the worst part of my day.
But you know, it isn’t all bad. It’s kind of nice in a weird way to get some time where I don’t ‘have’ to be doing anything in particular. Like I have an excuse to zone out or play on my phone without feeling too guilty. Part of me wants to make my bus trip more productive, to use commute time to learn some Hebrew or read some papers, but I know that I won’t be able to sustain this due to the general discomfort of being on a bus.
Maybe I’ll start to think seriously about moving out and moving closer to my lab and the other campus. I’m in no rush, mind you. It’s still cheaper to live where I am, I’m saving a nice portion of my stipend each month. So I guess I’ll just have to weigh the costs and benefits.
No featured pic this time, but as I’m flying off to Munich I’m sure there’ll be something picture-worthy I can share for next week.
I’ve been trying to validate the over-expression clones I generated. One of them seems positive, but the other is not working. Both clones actually of another cell line don’t seem to be behaving as I’d like them too. However, having that one cell line isn’t the most crucial, it is frustrating as the cell lines that I recovered from a liquid nitrogen also happened to not behave as expected either. I’ll think about whether it’s worth trying to regenerate this cell line or to cut my losses and to move on.
The CRISPR knock outs I’ve been working on are growing nicely (albeit slowly). I’m planning to grow them over the weekend so that I have enough cells to freeze both for regrowth and for harvesting DNA and further characterisation.
Purim celebrations this past week. Tel Aviv becomes a chaotic mess of people and music. So I went to Tel Aviv on the weekend with some friends to celebrate. I’m still not entirely sure the meaning behind the holiday. It has something to do with this guy, Haman, who wanted to kill all the Jews a long, long time ago. Somehow the Jews find out about this plot and instead kill Haman and his children and friends first. Modern day Jews celebrate their survival by dressing up and throwing a massive costume party. I don’t entirely understand the significance of all of this, but it was definitely a lot of fun.
This week has been one of my more productive weeks. Validating some cell lines I’ve been generating. One of them looks pretty good from a quick and dirty expression analysis. Will have to sequence and western blot to confirm. The CRISPR-KOs I’ve been creating are growing slowly but nicely. I hope I can freeze these guys before the end of the month.
I’ve also been able to draw a clearer picture as to what I need to focus on in the coming weeks/months. My PI and I have sketched out a working plan for experiments that need to be performed to tie up the loose ends for the project I’ve been continuing and to hopefully publish the paper in 5-6 months.
Featured pic is Esther, the Jewish queen who foiled Haman’s plot to kill the Jews.
Meant to make a post on the weekend but was travelling. I will be brief for this week’s entry as I’ll endeavour to write a lot more next week to cover what I’ve been up to recently.
There’s been a slew of terror attacks recently. Feel like I barely missed a few of them. A few days before a shooting in the West Bank, I was on a bus out of the settlements and passed through the entrance where the attack took place. And the night before I set off to Tel Aviv, the red alert siren was sounded in the wake of rocket strikes. I don’t feel particularly lucky per se, as the likelihood of being a victim of a terrorist attack (perhaps even in Israel) is lower than other, I suppose, ‘freak accidents’. But since these things are actually happening and there is a greater-than-zero chance that some violent incident will happen to me or people around me is somewhat disturbing.
But in other news, Purim is coming up soon. It’s a Jewish holiday where people dress up. Kind of like Halloween but less door-to-door solicitation.
What’s new this week?
Well, the spring semester here officially starts tomorrow (Sunday). I’m taking a course called “Computational Tools For Genetics”. The classes are twice a week and I’ll be learning programming in R and applying to these skills to problems in genetics using statistical methods. I’m pretty happy to get a chance to learn this in a structured environment. I’ll inevitably have to do some statistics sooner or later—when I finally get some data under my belt that is. I’ve also signed up for the Hebrew class this semester, I hope the next few months doesn’t get too demanding. Though I think I’m in need of a bit of a kick in the arse—for motivation/productivity and whatnot.
I’m itching for some data. At this point I just want to move as quickly as I can to wrap up the project from a previous PhD student I mentioned a while back. I have a bunch of ideas for prospective experiments that I want to start thinking seriously about and sketching out. But all of this relies on some data—any at all really— that I’ll need to compile. If this means repeating some experiments or resurrecting older lines of inquiry, so be it. I think I need to learn that sometimes it’s better to not always take the road less travelled.
In lieu of this I’m planning to culture some old cell lines and harvest RNA/DNA for sequencing/ChIP experiments. I need to talk to my supervisor to OK this and also if I can get some fresh antibodies. I also need to learn how to do Western blots.
The hygromycin selection I’ve been doing has yet to prove successful (or unsuccessful) all I can do is wait and keep on checking up on my cells.