# HG changeset patch # User Steve Losh # Date 1694233268 14400 # Node ID 677d49b2fda0fd0fc93b3547a2d2e094922b5ff4 # Parent 2be64da0672b0f9ebefe12d41665b5f509234956 Update diff -r 2be64da0672b -r 677d49b2fda0 README.markdown --- a/README.markdown Thu Sep 07 20:29:26 2023 -0400 +++ b/README.markdown Sat Sep 09 00:21:08 2023 -0400 @@ -1190,3 +1190,65 @@ Went to the poster session. Lots of stuff I don't understand, and a tiny bit that I do. +## 2023-09-08 + +HG545 this morning. + +Papers never say what *could* have gone wrong with what they did — you have to +just read between the lines and actively think about that (and what it would +have meant, and what you would have done if it did). + +Learned about nonsense-mediated decay: a mechanism where mRNA with premature +stop codons is degraded, instead of expressing a (probably truncated) protein. +Without this, if you have a mutation that creates a stop codon in the middle of +the gene, you would see truncated protein expressed. But because of NMD, the +mRNA is degraded and doesn't express the broken protein (as much). This is good +not only to reduce wasted translation, but because the truncated proteins can be +actively bad. + +One important control that was left out of the study where they wanted to find +where in the organism the target gene is being expressed: inject a probe with +GFP that intentionally shouldn't match *anything*, and expect it to show up +vaguely all over (or not at all). + +Another example covered during class: if you suspected a phenotype was caused by +a mutation in a promoter (instead of in an exon), how would you test this? +There were a couple of things folks came up with: + +* Could sequence the region in the mutant and wild-type population, compare to + see if the mutation segregates the two reliably. +* Old school: "reporter genes". I'm a bit fuzzy on this, but I think you insert + the promoter into a vector with some easily observable gene (e.g. luciferase, + a bioluminescent protein). Then you see if that product is expressed more or + less with the different variants of the promoter. This is a bit janky because + just yanking the promoter completely out of context can be problematic (e.g. + loses the chromatin structure around it, nearby enhancers/repressors, etc). +* Could use RNAseq to see if the mutants with the variants are producing more of + the RNA for that gene. +* Could use CHIPseq, if you know the transcription factors that bind to that + promoter. Fix, fragment, attach antibodies to the TFs, precipitate them out, + unfix, extract the DNA (all the remaining is whatever was bound to the + transcription factors), and then do the sequencing. You would expect to see + a larger signal if the mutation in the promoter is causing transcription + factors to be more likely to bind. + + +Got back and tried VPN'ing with the command-line client. It seemed to hang +after entering my password, but then I realized it had just silently tried to +2FA with my phone and I didn't notice. Trying again and being ready with Duo +let me log in, so I think I can probably ditch the webkit crap I installed for +the graphical thing. + +See lab notebook. + +Desktop machine wouldn't take input from my USB hub all of a sudden. Found some +bullshit in the logs, probably not worth debugging Yet More Linux Jank if I'm +just going to wipe this machine and install Debian on it soon anyway. Tried to +reboot and systemd hung at the end, so I just powercycled the damn thing. If +I could just have one single day where no computer broke for me, that would be +so nice. + +Flu shots are available, need to get one so PI doesn't get pinged all the time. + +Read for BS521 class. All still pretty basic. Cleaned up and turned in lab 0. +Finished homework 2 as well, just to get it out of the way.