Polishing MERFISH code for public release.
- take 140 gene library, create short fasta of just complete genes, run it through Launch OligoArray
- add parse OligoArray (as a function) into this script
- add assemble secondaries and target regions into as a function into this script (it mostly is a function)
- I think this should be a separate library building folder.
12:00 pm – 6:30 pm
(10:00 am – 12:00 pm working remotely on library).
- updated 15 kb en library to use F primer L6F5 with L6R2 instead of F1, which I already used in lib7
- ordered en 15 kb library along with probes for BB at 12K scale from CustomArray
- also sent MC our recent paper on chromatin imaging
Library name: L8_SI6.fasta
% 3′ P4-Alexa405, A647-Steven,
% 5′ FwdIndex, cy3-commonRT, TruncStevenSecondary, targetRegion, RevIndex
% 20-bp, 20 bp, 20 bp, 42 bp, 20 bp, total: 122
New libraries in consideration
- small MERFISH library to drosophila embryonic genes
- eve locus at 2 kb resolution
- cut locus at 5 kb resolution
- continued RT for 2kb resolution probe library against BX-C region
- updating protocol
- booked flight back from SFO to BOS on Sat Mar 5th.
- L7S (L7-secondary) arrived today
- S1-L7common fusion arrived today
- Hsuan offers to help. great!
9:05 am – 7:30 pm, 8:15 pm – 11:40 pm
- testing control L4E24 embryos. stains look great.
- compare to L7 embryos — no staining.
- ran series removing S2 with toe-hold 5 bp. Looks excellent.
- Gel tests
- try to anneal probe and secondary on bench, no cells. Test if still bound on gel.
- 5% PAGE gel. ran too long and gel was a bit too old
- smears suggest partially degraded RNA slowing down template to various degrees
- found the likely problem — 8 mM NaOH instead of 1 M NaOH in the RNA degradation step.
- re-ran digestion, re-ran probe clean-up
- re-ran hybridization
- S1rc + L7 common = GCGATGGTAGACGGCGTATGAATTCGGCAGAC GACCCGTCAGGATAGGCCCT
Microscope building prep
- requesting quotes from MPB, Nikon, Coherent, and Newport
- write microscope justification
- book flights back from San Fran
- diagnostic gel
- detailed labeling of samples