K.T.'s Gradblr

Thursday 1/5/23 - Life Update

My PhD journey came to an end last summer. I defended my thesis in June 2022, accepted a job offer at the end of July, also left my lab at the end of July, went on two vacations in August, and started my job as a medical device regulatory writer in September.

Life has been great after my thesis defense. It feels like a huge weight that I have been carrying around for years has been taken off. I feel lighter and taller, and the world seems so much brighter.

It was super nice to have 1.5 months of break to do absolutely nothing productive before I started my job, especially since I've felt time-starved for so long during my PhD. I had so much free time to sleep, muck around, and be as lazy as I wanted.

It's been 3.5 months since I started my job, and I've been really enjoy it a lot. It's a fully remote position for a fully remote company with employees all across the US, so everyone works at their normal time. My days end at 5pm, and I don't think about work again until the next day when I log in. I don't have email anxiety anymore. We actually have real flexibility regarding hours, so I have been starting my days a big earlier to go on a long midday walk with Percy. We have unlimited PTO, and I like seeing my boss and other employees actually taking advantage of it. I have not worked overtime once. This was my first time coming back from the holidays feeling actually refreshed and well-rested. We got so many days off, so I was very ready to get back to a routine. It's been very nice to work at a place that preaches work-life balance AND practices it.

I came across a journal entry from this time last year recently. This time last year, I felt like I was dragging a dead corpse to and from lab, trying to get all these experiments completed, working my regular 9-5 in addition to consecutive nights in lab. Last year me struggled so much mentally and emotionally with the workload and the exhaustion, and this year me really got to reap the benefits. Having finished with the PhD, I'm very grateful for my past self for persevering and pushing on when I was constantly daydreaming about dropping out or getting into minor accidents so I could be forced to rest (apparently a very common PhD daydream amongst my cohort...). I'm getting a lot of rest now, getting a lot of time to myself to spend however I want, either productively, creatively, leisurely, or lazily.

The only downside to my post-PhD life is my support system has moved away. We were all here for grad school, and now that grad school is over, everyone has scattered. I'm sticking around the area for the foreseeable future, and I'm working on rebuilding that system this year. Now that I have all the time to do it.

I started the actual writing for my thesis April 25th, sent it off to my committee May 30th, and had my thesis defense presentation on June 6th. That was 5 whole weeks to wrap up several data analysis and write an entire thesis from scratch (I had switched over to this project during COVID, after my thesis proposal, so the lit review I did for my thesis proposal unfortunately did not carry over). Here are my tips to get everything done.

Start earlier

Give yourself more time so you can have a more peaceful writing process and not be sweating and stressing for 1.5 months like me. However, I did have to write my thesis de novo in 5 weeks. If you have writing that you can put together into a thesis, that timeframe is much more doable. These writings can include thesis proposal, papers, etc.

Outline thesis chapters and create Google Doc for each chapter

This was done 1-2 months before the writing period started, in agreement with my advisor. Why to include in your thesis may be program dependent, so I would check with your admin person and advisor about this.

Using separate docs for the chapters is nice because it gives you little dopamine boosts as you finish one doc and move on to the next, as opposed to continuously working on the same doc the whole time and not getting the sense of accomplishment until the very end.

In addition, starting a new chapter is always a struggle for me as I try out new wordings and phrasing, and it’s kind of embarrassing to do with your advisor still on the same doc watching you do this. I like the separate Google Docs setup because I can send finished chapters to my advisor while I can have my initial struggles privately.

Use Google Doc as a Thesis Structuring Tool

I really like Google Doc because of the Outline function, which lets me have consistent heading formatting between the different chapters that can be combined at the end in Microsoft Word to generate a Table of Contents. I can also easily navigate through long chapters using this function.

If you have Google Doc through your institution, it’s possible that you also have the predictive text **feature, which I LOVE! It quickly learns the phrases you use as well as common phrases and suggests accordingly. I think it’s the most accurate and useful predictive text feature that I’ve used, but I also haven’t used too many.

Once all the chapters are finished and revised, I combine everything into a Word Doc, add in citations, add in figures, and generate the Table of Contents

Prioritize writing in the morning

Personal preference, but I felt like my brain was most clear in the morning and had the easiest time putting things together and synthesizing information from my reading.

This time also literature reading time, but the literature reading is done intentionally with the sole purpose to write and finish up chapters

Afternoons and evenings can be used for lower brain function tasks, like putting together figures, data analysis, adding citation, formatting, or low quality writing tasks (like the Materials and Methods section, writing captions for figures, etc).

Be intentional about breaks

This is so important! To make sure I take frequent breaks, I set a 1 hr timer on my phone and leave it outside of the room. That way, I have to leave my desk to turn it off. I did much better work when I took a 5 min break every hour (or 15 min break for every 2 hours worked).

During the breaks, I set another timer at my desk and take my break in a different room to separate work space from break space. I check social media, go for a walk, move my body, etc, and once the alarm goes off, I have to go back to the desk to turn it off.

Establish a ritual for writing and breaking

I did this because I wanted my brain to switch to writing mode when I did this particular ritual: light candle, place candle on top shelf, and ask Google to play lofi beats (it’s just whatever loft play Google wants to play that day so I can’t recommend a particular playlist).

When I take a break, the candle gets blown out, music is stopped, a timer on my desk gets set for the break time, and I step away from that room.

This helps me not stress about the thesis when I’m breaking and actually get to enjoy my break, and vice versa.

Outline extensively to give your writing a guide and directionality

This is a great way to show your advisor where you want to go with your writing without having a written draft

Don’t be afraid to be fluid with the outline. I have bullet points mixed with streams of consciousness, etc. It helps with the writing flow.

Write as much as you can without worrying about coherence, clarity, or quality.

Just type your thoughts as a stream of consciousness. Usually, they’ll come out dumb, incoherent, and cringey, and that’s all okay. All the first draft has to do is simply exist. Revising is 100% easier than synthesizing something from scratch, so get all of your thoughts out, dumb or not, and revise and organize later.

I used to sit there and make sure that every sentence is publication quality before moving on. There’s no time to write like that, it’s not efficient, and it interferes with my flow of thoughts. It’s QUANTITY over QUALITY when it comes to writing.

Accept that your thesis will not be the opus magnum you thought it would be

I was mentally paralyzed by how disappointing my thesis felt while I was writing it up, how it was so flawed and lacking and embarrassing that I produced work like this. I dreaded the idea of having to present this embarrassingly disappointing piece of work in front of my family and friends and them thinking, “she missed so much time with us for this p.o.s.???” Guess who else thought that about my thesis. No one. But coming to term with this allowed me to keep working on the thesis instead of being unable to move forward in fear of how inadequate it was in my mind.

Make peace with the idea that your thesis won’t be groundbreaking, Nobel prize winning working, and that’s okay. You just need a passing thesis, not a groundbreaking one, to graduate and move on with your life.

Limit your caffeine intake

I had multiple panic attacks a day until I severely reduced my caffeine intake. It’s a stressful time, and caffeine can really exacerbate that.

Take CBD

YMMV, but I took CBD several times a day, and it helped me immensely. I still felt the anxiety coming on but it was much easier to acknowledge the anxiety and keep working, rather than be consumed by it when I was not on CBD.

Have regular meetings/communications with your advisor

I had mine every other Friday for the last year before I defended. It helps a lot since it forces me to come prepared with stuff to show him, either in the forms of figures, outlines, etc.

During the thesis preparation phase, I would send off chapters as I finished writing them, and he would revise and send them back with comments.

Limit the number of decisions you have to make

I would recommend getting on some sort of meal plans that get delivered to you. We signed up for a meal kit plan, and my husband took over the cooking during this time. It was a huge blessing to just have food to eat when I was hungry without having to figure out what to order, how to make food, etc. My executive decision making capacity was in the negative during this time, so it was godsend to not have to make any decision outside of my thesis work.

Sayings that got me through the thesis preparation time:

Perfection is the enemy of progress

Eat the elephant one bite at a time

After putting it off for the last few weeks, I'd finally set my thesis defense date: Friday, May 27th at noon.

I've been doing ~50 hours/week for the past 4-5 weeks, and I'm exhausted. And the best part? It still feels like I'm so behind. It feels like I'm sprinting in place and uphill, but the floor is sliding down faster than I can run up.

I hate that my mice keep mating on days where I need to come in on the weekends to grab the litters 🙃 The past 4 weekends have been just litter processing 🙃🙃🙃

One thing that's been cheering me up: I've decided to use the academic "flexible" hours to my advantage. On Mondays and Wednesdays, my new schedule is:

9am-4pm: lab work

4pm-5pm: run in the park (while the sun is still out!)

5pm-5:45pm: quick dinner

6pm-9pm: back to lab

My schedule is way less depressing this way. I get to enjoy a brief moment of sunlight while not being dead exhausted, do something energizing, and return to lab feeling refreshed. And since I have a 3 hour chunk, I can take my time doing experiments until of trying to rush everything. Usually, me taking my time means finishing at 8:45pm, which means I can BE at home by 9pm, not trying to wrap up at 9pm.

When I started in my (then) brand new lab, I was brand new to mouse genetics myself. The idea of managing my own mouse colony was daunting. My PI was as helpful as he could be with various tips on how to keep the colony going, but when it came to how to organize all of this information, his trick was to have a photographic memory, which I don’t have. So, throughout my PhD, I worked on an organizational system that lets me keep track of my mouse breeding cages, all of my mice within the colony, and the trove of genotyping information associated with each animal, their progenitors, and their progeny. These are CRUCIAL components of the mouse colony that helps me maintain the mouse colony itself and obtain experimental animals. Obviously, there are other and better ways of maintaining a mouse colony, but this is just my way of doing it, founded by trials and errors.

Note: there are a lot of basics to mouse colony management that I won’t go into, like how to set up your breeding pairs, duration of gestation, when to wean, how to wean, when to replace breeders, etc. For further information, The Jackson Lab has amazing resources that can help you. For this post, I will only go over how to organize all of this information.

General Set Up

Cage card information

Google Sheet containing breeding cage information

Google Sheet containing holding cage information (recommended but optional)

Genotyping notebook/sheet/binder

Cage Card Information

I have a few types of cages in my colony with associated tasks:

breeding cages need to be checked once/twice a week for new litters and weaned when litter comes of age (red sticker)

male and female holding cages used to store studs and dams for future breedings to replenish the colony (blue and green sticker, respectively)

experimental cages are similar to holding cages but these animals are reserved for experiments and not breeding (yellow sticker)

timed mating cages are the same as breeding cages but need to be checked everyday before 10:30am for plugs (red+yellow sticker)

It’s extremely important to be able to differentiate these cages from one another. This way, I don’t waste time checking holding cages of only female or male mice for litters, accidentally miss new litters from breeding cages, accidentally set up a mating with animals I needed for experiments, or accidentally delay replenishing the colony by using future studs and dams for experiments.

I differentiate the different types of cages by a small colored sticker on the upper right side of the cage card. This way, I can quickly scan through my colony and grab the appropriate cages.

We also have help with the genotyping via a tech who brings tubes of DNA samples from new litters from the various cages along with associated papers with information pertaining to the parental cage, litter’s date of birth, and what to genotype for (more on this later). To make this process easier, I came up with my own naming system for the breeding cages. They’re usually a letter and number, followed by the unique number of the cage card.

Example: all of the cages for my Bmp matings will be B#. So for one particular cage, its full identifier is B6 (76411). I included the unique cage card number because I have mistakenly had two S10 cages before, and I was able to differentiate them only because I had also recorded the cage card numbers for both.

For information on the cage card, each mouse is identified by a toe number, genotype, parental cage, and date of birth (DOB). I ALWAYS include the parental cage and DOB every time I write down the animal’s information.

Real life reason: we started my first project with CRISPR’ed animals. Some of offsprings of these animals had mind-blowing phenotypes! Over time, we noticed that this phenotype can be seen in 50% of the mutant animals, and I unfortunately traced all of the affected animals to a few cages. The studs from these cages were all offspring of one of the founder mice. When we sequenced these studs, we found that these guys had the mutation we CRISPR’ed in, plus a few additional genetic modifications/additions in their genetic background (CRISPR artifacts are something NO ONE talks about until you mention it happens to you, then EVERYONE will share their experience 🙃). Because of my record keeping, we were able to eliminate animal samples from the affected cages. I did lose my phenotype but I’m grateful we did not publish data that would have to retracted down the line. What started off as fun record-keeping turned out to be a thesis-saving measure that I kept going forward.

TLDR: ALWAYS ALWAYS ALWAYS know where your mice came from. Throwing out some data points is sooo much better than throwing out all data points.

Google Sheet for Breeding Cages

I keep track of my breeding cages in a Google Sheet. Here is a snippet:

(note: there are two unique cage numbers for this cage because our vivarium recently transitioned from a card system with no unique identifiers to one with unique identifiers. For one without unique identifiers, I generated them based on the date the cage was setup using this template: YYMMDD(A/B/C/D/etc). I love the new cards since the numbers are shorter, but when you do see YYMMDD(A/B/C/D/etc), remember that is just the old unique ID).

In the Google Sheet, I record the animal’s toe number, genotype, parental cage (Lineage column), date of birth(DOB), and the cage’s date of mating (DOM). The date of mating helps weed out dud breeders and temper my expectation on when the cage should have new litters. The Age column is auto populated based on the DOB. When the age value matches or exceeds that of the value in the Replace At column, those cells will turn yellow, letting me know to switch out breeders.

I try to update this sheet once a month or at least once every two months. But this sheet has ALL of my current and past mating cages.

Google Sheet for Holding Cages

This sheet was a life-saver when I worked more with animals older than weaning age. At the time, I needed to differentiate between 30 days old animals (P30) that I can take brains from vs P60 animals that need to undergo a battery of behavior tests vs future breeders in holding cages.

Here is what some of the sheet looks like from March 2021. Mo-Seq is the behavioral test that I run on these animals, and a subset of these animals then gets transferred to a collaborating lab for further behavioral testing.

Currently, all of my animal samples are in the embryonic stage, so I’m not as up to date on this sheet as I should be, so sometimes I do have to wait longer for my stock of breeding females to replenish.

I do highly recommend keeping this sheet. I was able to show substantial loss of resources and PhD progress for a COVID relief fund at Rutgers thanks to my sheets, as I showed all the cages of aged mice I had saved for experiments, cages that were holding future breeders, etc, that had to be sacked when we went on lockdown, setting me back months. After we returned to the lab, it took my mouse colony 4-6 months to return to operating levels, and having a document to show that helped me secure that COVID relief fund.

Genotyping Notebook/Sheet/Binder

So far, I’ve gone over how to setup and differentiate cages of mice that I know the genotype of, so let’s go into HOW I get the genotypes and how to keep a record of it via my genotyping notebook/sheet/binder system.

I check my breeding cages (marked by red sticker) once a week for newborn litters. At this point, I’m fairly comfortable estimating their age, but if you’re not, consider checking twice a week and/or refer to this chart. If I see new litters, I’ll record their DOB, note which alleles needed to be genotyped, and mark the cage for our tech, who then collects DNA samples from these pups, fills out my genotyping sheet (see below) with the alleles I’ve marked, and brings them upstairs for us to perform the genotyping.

Above is what the sheet looks like when we receive it, along with the DNA sample. It has the parental cage information (B4 - 210811A), the alleles I need to genotype for, the number of pups, and their DOB.

We then perform the necessary PCRs and run out the gels, which are all recorded in specific genotyping notebooks (GN#) that are separate from my experimental lab notebook (LN#).

This is what the gels look like in the genotyping notebook. From here, I interpret and record the results and locations of the gels. To interpret the gels, I first check to see whether the control samples from each type of alleles are clear and of expected length. If the control bands are not clear or not expected, I go straight into repeating the PCR. If they are good to go, only then do I check for the appearance of the sample bands.

Notice how there are control samples for each type of allele that was amplified (Allele 3’s controls are cut off). The “-” control denotes a water sample AKA negative DNA, meant to detect DNA contamination in the PCR master mix for that specific allele. If we get a band here, that means that there was DNA contamination, the gel is not reliable, and genotyping has to be repeated with fresh water, primers, and PCR master mix.

For the W and M wells, we use DNA from previous genotyped samples that are known to be either wildtype or mutantfor this allele, respectively. This confirms that the correct primers were used to amplify this specific allele (are the wildtype and mutant bands the right sizes?), and that the amplification proceeded correctly for this batch of samples (are the bands bright or faint? If the PCR machine was unexpectedly shut off, then seemingly negative samples that are faint may be positive but didn’t have the chance to amplify adequately). The W and M controls are nice to have for genotyping where I directly amplify alleles and run out the products on the gel. However, they are CRUCIAL if I need perform a restriction enzyme (RE) digest after the PCR amplification, which can detect whether the RE incubation condition was sufficient. Control bands looking like they should is a great indicator that the PCRs were performed correctly and the genotyping results are reliable.

Since the control and sample bands look great for all three alleles, I record the results and the location of the gels. For example, the gel for Allele 1 can be found on page 116 of Genotyping Notebook #3.

When this litter reaches 20 days of age, I bring this sheet downstairs to help me separate out animals with our desired alleles from the ones without. Once they are weaned, I either write “weaned (date)” or check off the wean by date I’ve given myself (see above).

After weaning, this sheet goes into a large binder where I keep all of our genotyping information, organized by most recent DOB in the front.

Example: I set up a mating cage with Mouse #1 (from cage B4 (210811A) DOB 11/2/21), who is heterozygous for Allele 1 (let’s say positive for Allele 1), with another mouse (from cage XX (YYMMDDA) DOB MM/DD/YY), who is homozygous WT from Allele 1 (negative for Allele 1). However, the past two litters from this cage had zero offspring who is positive for Allele 1, when Mendelian genetic predicts that 25% of offspring would be. To see why this is the case, I check the stud and dams’ information and find out that I had accidentally set up Mouse #2 (from cage B4 (210811A) DOB 11/2/21) instead of Mouse #1. Since this would be 2 months from their DOB, I don’t have Mouse #2’s genetic info off the top of my head, so I search for the genotyping sheet for B4 (210811A) DOB 11/2/21. Lo and behold, Mouse #2 is also negative for Allele 1, explaining why all of the offsprings from this mating were also negative for Allele 1. If I did set up Mouse #1 instead, then luck wasn’t on my side but I could rest assured that an Allele 1 positive animal would eventually come from this mating.

Are there better and less convoluted way of doing this? Definitely! This is just a system I use that is most intuitive for how my brain works. The Google sheets help keep track of breeding information and plan out experiments, in addition to keeping track of the animal information. The strength of genotyping notebook/sheet/binder system comes from the ability to trace animal’s genotyping information and relate that to its progenitors and progeny.

Once I'm done with my PhD, I would like to not do bench work anymore. Bench work isn't linear. You could put in so much effort and hours and still end up with zero data, which is not an unfamiliar feeling for me. I remember beating myself up over failed cloning experiments my first year in lab, my inner voice telling me I wasn't working hard enough, wasn't thinking about the problem enough, and if I would just try it again (and again and again and again for the entire summer), I would finally get the cloning product that we needed to move forward with the project. I never got it to work, so I had exactly zero to show for all of the late nights, weekends, and holidays spent in lab.

I woke up at 6am today and went through my whole morning routine. Felt great! Got to lab at 9am and got down to business.

And THEN I found out that the mouse that I was supposed to give me E14 pups wasn't actually pregnant, just fat...so that was a waste of 14 days + all the days I spent checking for plugs...

And THEN I also found out that the experiment I stayed late for yesterday didn't work lmao 🙃 🙃 🙃

I was going to do some more experiments in the evening while imaging, but I've scrapped that since today just feels very unlucky and I don't want to jinx my future self.

On a more positive note:

My grad program had free cupcakes today! The professor in charge actually went to my building so the grad students here could actually participate for once since we're quite a drive from the main campus.

It was a beautiful day, so I went running with Percy at the local park.