9:10 am – 1:05 am
MERFISH data analysis
- Lots of little coding changes
Changes to LoadMERFISHdax
- Handling collections of info files
- cell arrays of structures are hard to index
- these should just be multi-element structures
- rewriting all info file stacks to conform.
New analysis
- running InsightM on
\\morgan\MorganData2\MERFISHdata\150715_L16Test4\5uM_restain\Laplace data - running daoSTORM on
\\morgan\MorganData2\MERFISHdata\150715_L16Test4\5uM_restain\‘Raw’ data (post-bleach subtracted data)
additional descriptions and data
- see my Evernote notebook entry for today (team shared lab-notebook)
Some additional Coding notes
- wrote
MovieToMlist - can get running fitting algorithm directly on an image in matlab (i.e. a 3D matrix)
- can also pass parameters
mList = MovieToMlist(dax(:,:,1),'daostormPars',{'threshold','bkd'},'daostormValues',{'100','0'});
RNAi experiments
RNA cleanup
remove template DNA
- first, DNase digestion of template following NEB (2x since we doubled the RNA synthesis volume) protocol
- “To remove template DNA, add 30 μl nuclease-free water to each 20 μl reaction, followed by 2 μl of DNase I (RNase-free), mix and incubate for 15 minutes at 37°C.”
cleanup with Agincourt (Beckmann-Coulter) RNA-clean XP beads
- online protocol
- Add 1.8 volumes of beads to sample in 1.7 mL tubes (min sample volume 100 uL + 180 beads)
- Mix by pipetting, incubate 5-20 minutes at RT (not on magnet)
- Separate on magnet until clear
- discard solution
- rinse 3x in 70% ethanol (500 uL – 1 mL)
- let dry 10 minutes
- Elute in >30 uL RNase free water
Quality checks
- RNA concentrations all in the ~1000 ng/uL – 1500 ng/uL range according to nanodrop
- running on 5% (2 month expired) TBE Urea PAGE gel
- also forgot to remove tape. Still getting substantial product trapped in wells. Both RNA and DNA ladder look smeary. (somewhat old DNA ladder, maybe it is also a bit degraded). Still, hard to explain the larger than band smears based on degradation when running a denaturing gel.
new RNAi set up with new dsRNA
- SCM single
- SCE single
- Su(Z)12 single
- Pc single
- Esc single
- Pc, Ph-p, Ph-d, Esc, Scm
- Pc, Esc, Scm (Forgot to make more Psc RNA!!!)
- water control
New RNAi primers
- new Psc (PRC1 + dRAF)
- new SCE/dRING (PRC1 + dRAF)
- new Esc (PRC2)
- new Su(Z)12 (PRC2)
- E(Z) (PRC2)
- N55 (PRC2) [no sequences]
- ordered new primers with T7 to make more RNAi
More dsRNA synthesis
- PCR order (8 tubes)
- Pc v2, Pc v2, Psc, Psc, Esc, Esc, Scm, Scm.
Data analysis
- new images of more BX-C cells don’t look that changed (small offset peak, median is still on the original median). Suspect some effects of not excluding single alleles.
Cell culture
- my BG3 cells might be contaminated. (cells dying)
- probably an issue with not filter sterilizing the new culture media with the new insulin?
- hopefully it’s not an issue with the new FBS, this would affect my RNAi as well
- maybe I got the insulin concentration wrong?
- don’t see any signs of growth in the media. I’ll leave it out for a while and see what happens
- restarted frozen stock and transferred to the original Schneider’s in FBS.
- so far these cells look happy-ish. We’ll see how they’re doing tomorrow
- (shouldn’t have killed the stock culture yet, but I guess can always re-order)
- ordered more insulin (got the recommended human insulin in solution instead of the lyopholized cow insulin). Maybe this is what killed my cells. Hope the new stuff arrives.