- AP patterning (13)
- Chromatin (56)
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- Microscopy (77)
- probe and plasmid building (57)
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- Research Planning (65)
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- snail patterning (40)
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Tagsanalysis bcd cell culture cell labeling cell staining chromatin cloning coding communication confocal data analysis embryo collection embryo fixation embryo labeling embryo staining figures fly work genomics hb image analysis image processing images in situs Library2 literature making antibodies meeting modeling MP12 mRNA counting Ph planning presentation probe making result sectioning section staining shadow enhancers sna snail staining STORM STORM analysis troubleshooting writing
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- Sequencing analysis
- plots of coverage uniformity per probe set
- plots of failed probe vs. purity using new indexed method (or maybe just a length cut-off)
- Ph Project
- restart simulations
- start building library 4 (on Monet)
- start building library 5 (on Odyssey)
- Chromatin imaging
- Start STORM fitting new regions:
- (D08 and D10 from STORM4, D11 and F6 from STORM2)
- ChromatinCropper of new regions
- Chromatin Library 3
- cut up G10 into subsections
- start building library
- Finish O/N STORM on STORM2 of D11
- move Kc cells from -20C to LN2 (11am)
- Relaunched Ph simulations with capping model parameters
- Building Fasta file of all transcribed genes for project2 cell line.
- Restart Fasta file building for genomics
- removing GPU from STORMrender
- not compatible with CUDA 5.5
- not restorable after CUDA update
- not distributable / not multiplatform
- Computer reboot terminated building gene list, around 60% complete
- Relaunched on Tuck, with directions to write genes to fasta file as it goes. This way if it gets aborted we can restart from where it left off.
- may take 2 – 3 more days running on Tuck.
- Too much memory used. Cancelled run on Tuck (1.2% complete).
- Restarted on Monet
- Monet rebooted to install new CUDA
- Relaunched analysis. (CUDA openMM failed).
- Finish O/N STORM of D08
- Set up O/N STORM on next sample on STORM2: E11 (50kb Green, scattered repeats).
Chromatin region analysis
- Remake G5, test in new modENCODE sequenced Kc cells
- Regions to test sequencing results:
- G4 (failed repeatedly)
- F3, F4, F5 (haven’t made succesfully)
- Note: saved index is reverse-complement of the last 20 bp
- chop of indices and keep sequence in between, align that to sub-library.
- Library purity under-estimated: reads with just outside primers all reported as off target reads.
- Reads from E06 sublibrary that have long off target inserts (>30 bp) have the E06 primer cross hybridized most frequently to something that vaguely resembles the T7 primer: note the ‘GGG’/'CCC’ and ‘ATTA’/'TAAT’ regions.
- all told there are a bit less than 17,000 of these long insert off target sequences.
- cutting to 10 bp or longer inserts the motif is less pronounced. (373,000 sequences)
- So if I restrict my analysis to reads that have 10+ bp of sequence in between the common primer and the index primer as you suggested, I can validate that the short reads largely do result from mispriming on other sublibraries. (This is easily demonstrated by aligning the non-primer sequence of 10+bps back to my library as we discussed). I take this as good news because it library cross-talk should be correctable by dual indexing. (When I found a lot of reads with almost nothing in-between I was concerned that additional problems aside from sublibrary cross-talk plagued the short libraries, which might not be correctable by dual indexing).
- This lets me return to the motif question: Combining all the reads which result from mispriming of the index 10bp or more away from the end of the common site, (10 bp+ of sequence gives me a little more confidence that the reference has been mapped correctly) I get a weak motif (see attached). Doesn’t seem to suggest a strong sequence bias. If I look at the random nucleotide sequence added on in the adapter to help control for PCR amplification bias skewing the sequence the diversity is very high. So what motif like nature there is to this logo is probably not an artifact of the sequencing prep PCRs.
New primer screening
- oligoprop all index-T7 fusions
- no correlation between difficult to get spots and T7/index dimer hairpins
- Test new short probes (8 hybes)
- On deck: new RNA stains
- On deck: restain G5 (with RNase)
9:10a – 11:45p
- Lab Meeting: private notes
- Journal Club: notes
- Mentoring Meeting
- Bogdan will explore length dependence in Blue domains
- BXC ~ 1900 nucleosomes — feasible scale for simulation
- Bogdan attempting to get GPU accelerated openMM working on Cajal
- Moved to Monet for safety. Unable to install easily.
- NVIDIA CUDA 5.5 breaks compatability with 4.1, previous GPU acceleration for STORMrender
- Attempting to reverse by uninstalling CUDA 5.5
- OpenMM 5.1 is available on Odyssey.
- CUDA not working on GPU still. TdrDelay had disappeared. Wrote new TdrDelay DWORD. System needs reboot for changes to be applied. Wait until after genome processing is done.
- Passaged cells
- Froze 10 vials of Kc cells (combined 2 confluent 75 cm^2 chambers).
- Tomorrow remember to move cells to LN2.
- Building Fasta file from cufflinks GTF output
- Need to ID most expressed isoform for each gene
- Need to find the list of introns / utrs etc corresponding to that isoform, and the strand orientation
- Go to the genome fasta files by chromosome and grab the corresponding sequences. Splice them all together and reverse complement if necessary.
- info on GTF format.
- Running conversion overnight
- Finished O/N STORM imaging of D10
- D08 (50kb Green) seems to stain alright. Not especially bright, background a bit high, but working.
- running O/N STORM imaging of D08
- Bogdan imaging on STORM2
- notes on STORM2: slider tucked in dark box of cMOS camera is now the control to switch between camera ports on STORM2. Be careful not to turn the slider!
- Moving the slider does require backport alignment to be repeated. (and beads to be re-imaged!)
Fluorescent proteins as a scaffold for cell-specific gene manipulation
- Lots of GFP lines
- not a lot of Cre / lox or Gal4 lines (etc) for manipulating gene expression.
- camilid VHH antigen binding domain (‘nanobody’).
- Previous work: different nanobodies against GFP change it’s fluorescent intensity (‘enhancers’ and ‘minimizers’)
- screen for GFP nanobodies
- recruit VP16 to target genes in a GFP dependent manner.
- why not genetically encode, reporter gene and nano-bodies, then cross this to a GFP mouse line.
- Concern: low level GFP still respond
- GFP lines not very specific — have a high expressing cell type and a low expressing cell type.
10:00a – 11:00a, 3:00p – 5:00p, 7:00p-12:30p
Deep Seq Analysis
- Analyze distribution along probe region of copy number variation
- Analyze sequence dependence of copy number variation
- If a sequence contains a deletion, do we also have that same sequence without a deletion?
- Off target binding motif analysis?
Hao Lib4 prep
- Analyze Isoform data
- image G4 cells new probes
- Cufflinks data analysis
- Several key genes skipped as HIDATA.
[Default is 1000000]. This is too small for GAPDH
- downloading human chromosomes as separate fasta files: ftp://ftp.ensembl.org/pub/release-73/fasta/homo_sapiens/dna/.
- Writing fasta builder to construct fasta file with only most highly expressed isoform.
- finish overnight STORM run E10rna
- Test STORM of G4. FAILED
- Test STORM of F3. FAILED
- STORM of D10. Not all nuc have obvious stains. Look more like an RNA labeling. No idea why, had good denaturing…
- Lack of change in Ph Model in response to PhM levels under diff model is Expected under the limited binding / K27 of course. Obviously if it doesn’t increase with more wt Ph it won’t increase with more mutant Ph.
- Really should move this simulations onto Odyssey. Write it such that each level/round is run on a different core and compiled at the end.
10:00a – 11:00p
- Helping Guipeng and Ruobo get set up on STORM2.
- prepping cells for hybridizaton.
- last row (4 coverslips) forgot to add last rinse of PBS. Cells dried out. Coverslips tossed.
- cells into formamide, ready for staining.
- Samples to test: D8, D10, E3, E11, F3, G4
- All stained, incubating at 47C, ~7pm
Library / Deep Seq analysis
- Rebuilt Lib2 fasta with indexed headers (Library2idx.fasta)
- Re-running bowtie2 on on indexed Lib2 fasta
- still running at 10pm
- Cajal has 12 memory slots and 6x 4Gb currently on board. Could add 4×4 Gb more from Monet upgrade.
- Add ROI analysis to STORMfinder
- To add to Insight, modify .ini file to contain in ‘Image’ field the following lines: ROI Valid=1 ROI_x0=0 ROI_x1=256 ROI_y0=0 ROI_y1=256
- Sync Monet, Tuck and Cajal Git to use ZhuangLab/storm-analysis git repo as the DaoSTORM location (this will remain updated by hazen).
- test insight ROI on STORMfinder. Seems to work excellent
- Investigating new ROI in DaoSTORM
- successfully integrated ROI specification in STORMfinder / matlab-storm repo.
- Box4 went unresponsive during final stages of analysis of G9 data. Several daoSTORM runs failed, possibly corrupted.
- Running G1 data on Monet using new ROI finder.
- set up overnight STORM of E10rna stained cells in V marked region.
- V clearly visible in 10x image but not in 100x image.
- hopefully the large cells act as landmarks. Could have tried imaging more beads.
- working on slides for meeting with Ting
- tentatively scheduled for Dec 3, 2pm.
- extra F6 data set on STORM2. Is this really F6?! Why would I image F6 on 11/08, I had plenty of F6 before. Check with Bogdan.
9:45a – 11:50a (remotely)
12:00p – 7:10p
- A549 whole cell data finished running cufflinks. Bottom half of genes are empty / all have zero FKPM (chr12 on).
- attempting to run cufflinks 2.1 centos6 version on cytosol BAM file directly
- Run direct on BAM fails.
- SAM files too big to run from home scratch directory. Moving to Zhuang lab directory.
- WinSCP onto login.rc.fas.harvard.edu
- calculating size of zhuang_lab (max is 1Tb, hopefully there is enough space here to convert all the BAM to SAM so we can run cufflinks even though the header files from ENCODE are crap).
- Yari’s folder here is empty and mine has only 12 Gb, (now more like 100), so we should be fine without blowing this cap yet.
- Or we could if WinSCP / odyssey connection wouldn’t keep on dropping me. This is very annoying.
- WinSCP is EXTREMELY (impossibly?) slow at deleting large numbers of files from server (e.g. 3 entries per gene in the human genome). Connection stays at 0% deleting forever and then hangs.
- SAM2BAM on A549
NODE_FAIL. what’s this mean?
- no effect of PhM concentration at all in koff model. Is this a perfectly balanced composition? Weaker binding but higher conc?
- no, I think it’s just the capping model is effective at quenching binding at pretty low concentrations
- reality is probably a hybrid condition, a bit weaker association as well as lower
- if 1st two objective mirrors won’t align optics, use third mirror up down.
- Realigned back optics
- Reset back apeture for coarse alignment so it is in register with the aligned beam (previously was not).
- Replaced missing screw on Quadview
- Realigned Quad view following directions
- STORM2 and quadview now algined
- imaged beads
- imaged negative control for D12, looks excellent. Imaged with crosslinked beads. Beads much too sparse. Try 1:500 next time?
- Etching works best with VERY LIGHT TOUCH! deeply scoring gives ugly marks AND causes coverslip to break! Tragedy!
- D12 RNA sample destroyed. Try again tomorrow with E10 sample
- G9 data now running on Tuck (should have done this earlier!)
- G1 data now running splitdax (and conflicting badly with the disk write to the G9 data, but oh well).
- Cells growing excellently.
- Main original 3 flasks will need passage again tomorrow — move to large flask, grow up densly in prep for freezing.
- Split off separate culture flask for Bogdan with separate stock solution. Need to order new stock vial tomorrow.
- Plated and fixed 12 coverslips of cells for Bogdan and myself each.
- Take these through the prep protocol tomorrow.