9:00 am – ?

Getting Drosophila expression data

• Failed to find assembled FPKM data
• downloading 0-4hr data sets from modENCODE
  • What’s with the big time window. There must be better these?
• need a GTF file for cufflinks to parse the SAM data
• running cufflinks: errors
  • need to convert SAM to BAM
  • downloaded GTF from UCSC refFlat.txt – this gives errors
  • downloaded R5 data from ENSEMBL (release 5 is getting hard to find now everything defaults to release 6) ftp://ftp.ensembl.org/pub/release-77/gtf/drosophila_melanogaster
  • RC Odyssey is very slow today. Was giving lots of acct errors too: server not responding. And my jobs stay in Pending purgatory for an hour. Better than all day I suppose.

Probe Design

• finished design of probes, check probe length
• still have hundreds with good length
• computed sum FPKM from slicing data (this data is truly blastoderm — Hox genes and Segment polarity genes inv / en are cold off. Would be lovely to have some later embryos. Shame the modENCODE data is so broad. How about some single embryos at close timepoints?
• downloaded blastoderm single embryo data from Susan Lott’s paper
  
  Hox Activation
  

Ordering control probes

• submitted orders to Alec for BioSearch / Stellaris probes for Kr and tll as positive controls.

9:00 am – 6:00 pm

Meetings

• discussion with patent lawyer
• discussion with MERFISH team

Computer troubleshooting

• User Profile Disks might be problem with user session management: http://www.virtualizationadmin.com/articles-tutorials/vdi-articles/general/working-with-user-profile-disks-on-session-based-desktop-deployments.html
• disabled User Profile Disks (these were not enabled on CAJAL but were enabled on MORGAN when MORGAN started deleting user account data).
• re-installing local code database on my MORGAN account
• running OligoArray with 20mers 66C – 76C for all IMR90 transcriptome (37,442 genes)
• launches about as fast as when running on Cajal, but uses no CPU (maybe saving over the network uses more CPU?)

Tissue section prep

• Ultratome booked today and tomorrow. I booked Friday after lab meeting

Presentation Prep

• working on slides for journal-club presentation on past 2 years of Hi-C.

Fly patterning

• Created Fasta file of all 547 annotated patterning genes (has all the major players), selected longest isoform for each.
• Playing with embryo section-seq data
  

9:00 am – 6:00 pm

Literature

• Gomez-Diaz and Corces 3D genome architecture review

Experiment Planning

Drosophila Patterning screen

• started new Projects folder TF interactions
• Downloaded list of 1000 annotated sequence-specific TFs (GO annotation) from FlyBase. This list has substantial number of non-melanogaster genes to be removed.
• Extract gene IDs, Extract sequences
• Flybase exon download extracts exons of different lengths but mostly overlapping sequence as different exons.
• use transcripts instead, just select longest transcript

Plan

• order tiling probes from BioSearch (30 probes) against two select genes

Potential cover art ideas

• http://eoimages.gsfc.nasa.gov/images/imagerecords/78000/78674/london_lights_2012087_lrg.jpg

Chromatin imaging

• move embryos into 30% sucrose for cutting

9:00 am – 7:30 pm

Finalizing draft of chromatin manuscript

• revised gene expression panels in figure 1
• added additional references to text
• wrote figure captions

Additional references for chromatin literature

P Geyer

• chromatin organization and function (spatial positioning effects) 2011 http://www.sciencedirect.com/science/article/pii/S0955067411000196
• chromatin organization and function (editorial, histone code and 3D genome organization) 2008 http://ac.els-cdn.com/S0027510708002522/1-s2.0-S0027510708002522-main.pdf?_tid=abb4d7a8-d194-11e4-906b-00000aacb361&acdnat=1427139899_12a077eb9fafec4dbe28b479c17def4b
• Insulators review (2003): http://www.sciencedirect.com/science/article/pii/S0955067403000395#

G Bosco

• Condensins and chromatin organization: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3927828/pdf/nihms528141.pdf

G Karpen

• Nucleus (editorial intro) http://www.sciencedirect.com/science/article/pii/S0955067414000581#. Discusses 3D genome organization hypothesis

Other relevant chromatin literature

• continue reading up recent Hi-C publications:
• van Bortle 2014 Genome Biology on Insulator function
• Seitan 2013 on Cohesin
• Schoenfelder 2015 on Promoter-enhancer interactions by promoter capture Hi-C

10:00 am – 12:45 am

Chromatin Project

wrote code FindDataFiles to return a list of all the folders on data drives. These can be passed to StringFind to find

Figure 1

• finalizing layout
• added gene expression bar-graphs (doesn’t look that good)

Figure 2

• added quantifications
• explored alternative panel layout
• reverted
• modified internal scaling to move legends outside. Still need to update printing of these.

Figure 3

• wrote new code to process new domain data.
• manually rescreened all data. saved as new interactData.matb in the Index folder in Chromatin\Data
• minor screwup saving frac_irINred and frac_redINir into interactData structure
• not a real problem as these can be recomputed from the raw data also saved in dist_ir2red and v.v. See Fig 4 assemble script.

Figure 4

• started new version of the figure script. cleaned up the code a little bit.
• fixed schematic images as discussed.
• very short black inside a very large polymer don’t coil back on self enough. Need larger black. Trying this now.

Next steps:

• Figures probably in good enough shape to return to text…

9:00 am – 11:30 pm

Chromatin Analysis

• re-analyzed C02 data (originally taken on STORM1, not the best data, background separation a bit difficult).
• looking an Kc CNV
  • this genome is a bit of a mess (not sure how much noise is in this data either), but spot checking regions none seem especially or broadly out of average. Bxd is apparently over-replicated.
  • tried to compare to clone8, but the modEncode link is broken

Exploring metrics

File names explain metrics (click to see). See also y-axis label

To do

• CoV of pdist matrix
• this looks promising:

9:15 am – 7:00 pm

Chromatin Analysis

• analyzed up through dot 2 (29) of new D08 data (through dot 64). Looks great.
• analyzing fractal dimension parameters — computing for all composite Flists (this is very slow so hopefully I’m computing it right!)

Manuscript Prep

• laid out fig4

10:15 am – 10:25 pm

Meeting with Guy Nir

• discuss TAD organization
• discuss STORM buffers (see lab protocols)
• discussed STORM analysis – see ZhuangLab Github and tutorials
• discussed converting Nikon format to matlab format.
  • Jeff says Nikon has a dax converter available upon request

Computer issues

• Jeff C. fixed firewall issue
• ran windows update (see if the re-introduced firewall blocking server detection of my local computer again)
• to fix, in advanced firewall options, select files and folders

Chromatin

• running export of 2 color data to multi-color panels (for some reason this froze last night)
• analyzing unprocessed black 2 color data. D10-6, D11-7. Looks okay. Didn’t have same-day beads, slight offset I think.
• analyzed D11-6 D12-7 data (these are better)
• analyzing D08 data. This looks good. Got 30 cells done. Should compare to rest of data to see how it looks.

Coding

• fixed bug in Translate image
• updated substantially PlotBlobExample3D and Plot2Color3Dchromatin in Chromatin\Code\Functions.
• we really want this built into the Cropper.
• added new options to adjust contrast of conventional images being used for auto-correlation image alignment (this works much better).

9:00 am – 12:15 am

Little things

• # move cells to LN2 storage
• Fixed MORGAN Remote Desktop (see post with today’s date)

Project 2

• unofficial report – reviewers satisfied with manuscript revisions
• project 2 meeting
• minor text adjustments to project 2

Chromatin

• discussion with Bogdan about ChIP
• exporting panels of all 2 color data (I like these)
  • a lot of these will probably want some 3D spinning to get an optimal view
  • my 3 viewer seems to chose an arbitrary angle to project. (some are clearly x-z instead of x-y orientations)
  • some issues with size, increased FOV to capture some of the whole two color domains. hopefully export with scalebars will fix this.
  • should try with the color mixing in addition to the color transparency approach (currently doing color transparancy).
  • some of these views probably should get rejected as likely junk. Most are reasonable though.
• fixed 1 color data report to export up to 75 images (instead of maxing out at 25)
  • still need to rerun this.

Figure adjustments

• automated ChIP domain annotations: see F2_ChromatinImages150311.m in Chromatin