9:35 A – 7:00P, 8:20P – 11:50P
STORM
- chromewarp 3D has much more trouble vertically matching beads when we take the same field for each z. Need to change this. 1 manual edit of the beadrun.xml file ought to do it.
- wrote short matlab script for writing beadrun.xml files. (too long for manual edit).
- Could also try rewriting code to do ensure that matches are only done between ‘passes’? Good way to get better fit on previous data. Maybe not worth time at the moment.
- reshoot beads with new beadrun.xml file (15 regions, z sections 600 to -600 in 100 nm steps)
- reshoot again with longer delay (x,y and z stage all need a little time to move and settle down).
Doory Lab meeting, 10A-1:20 P
Oliver Bell Seminar Highlights (2P – 3P)
- Add binding sites upstream of Oct4, add small molecule mediator (Rap or ABA) mediates binding. Replace exon 1 with GFP.
- recruiting HP1-alpha silencing locus in ES cells (normally H3K4me3, actively transcribed, GFP expressing),
- DNA methylation doesn’t appear until long after HP1-alpha is established.
- washing out HP1-alpha gene is reactivated. Happens slower if DNA methylated but still happens. (‘not epigenetically inhereted’, activators still present).
- recruiting VP16 in silent MEF cells (H3K27me3, H3K9me3, silent), activates (12% of cells).
- Recruiting HP1-alpha to VP16 activated MEF cells silences them. Remove both, stays silent (epigenetically inherted for 10+ generations).
- Recruiting EZH2 -> no effect in ES cells, H3K27me3 in MEF cells.
- Narrow domain of spreading in MEF cells, larger domain of spreading in ES cells.
Meeting with N Francis (5P-6P)
- Pc repression of actively transcribed genes in cell culture (Psc, cyclin B)
- Initiation of Pc repression: hand-off from gap repression
- Full list of interesting tracks can’t be uploaded to UCSC in one shot. Attempting smaller list of PcG factors
- still errors:
Error File ‘Pc_EZ_Pho_CTCF_Pol2.wig’ – track load error (track name=’ct_S2PolII_1179′):
Expecting four words for bed format data at line 2845508, found 1
WARNING: Exceeded chr4 size 17445811 > 1351857. dropping 16093955 data point(s)
WARNING: Exceeded chr4 size 17446835 > 1351857. dropping 16094979 data point(s) - loading separately gives no errors…
- still errors:
Francis lab journal club (6p-7P): Nogales lab PRC2 paper.
Fly work
- PcG mutant stocks arrived, label add to stock collection.
- 2x HbZ balancers starting to emerge — collect and separate males / females / virgins (will sort and cross later).
- Move 2x HbZ crosses to 18C for further virgin collection
- MTD x Su(Z)12 F1s are emerging –> let self, pool all F1s in collection cages.
- Pc1 s MTD F1s close to emerging.
Snail
- Revisions back for CR.
- Fix minor text issue for reviewer 1
- To do for reviewer 3
- # mRNA counts per cell in double labeled cells
- # mRNA counts per cell in similarly age
- # clarify methods section discussion of assigning cells to nuclei.
- Set up PCR for Espl deletion screening
- PCR order: Espl-DNA + E1, E2, E1&2, sim(all), sim-DNA + E1, E2, E1&2,sim(all)
- E1 = Primers EL3-EL5+ER3-ER7
- E2 = Primers EL6-EL8+ER8-ER13
- sim(all) = Primers simDF2-4 and simDR2-3
- 12 min elongation, 65C annealing (should repeat with a 26 min elongation).
To do
- Analyze images from recent STORM run
- collect beads on STORM2
- phenol extractions of MP05 MP10 (save for in parallel with Espl,sim recombinants?)
- RNA FISH in cryo sections?