--- 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.