Protected: Ph conc effects

This content is password protected. To view it please enter your password below:

Posted in Chromatin | Tagged | Comments Off

03/30/15

9:30 am – 10:15 pm

Manuscripts

Ph Project

  • reply to Ajaz and Nicole about modeling work
  • send back comments on plan to add model figure
  • should try to compute fraction of localizations/molecules in clusters over 30 nm as a function of total Ph concentration for all types. Try this later…

Oligo Secondaries project with Wu lab

  • final comments for last round review / response to reviewers (hopefully) sent to BB

Chromatin scaling manuscript

  • working on introduction

Discussions on HiC

  • Questions for Max and Geoff:
  • evidence of domain switching (looking at the large scale, off diagonal interaction preferences, change a domain from repressed to active (in a different cell line or by stimulation) and show that it switches from compartment B to compartment A).
  • supposedly this is clear from Dixon 2012 and Nora 2012 data

Other notes

  • 3C / 4C / 5C hard to have a simple and accurate normalization — not clear what’s visibility vs. contacts
  • linker domains hard to formalize
  • domains follow epigenetic marks but not dependent on them
  • CTCF / cohesin not yet shown to have a role in metazoan genomes but maybe that’s an assay problem: sufficient time for domains to re-equilibrate after knockdown? sufficient knockdown?
  • supposedly removing a CTCF (or cohesin) loader is sufficient to cause more substantial changes?

improving 3D data

  • looking for better correction factor (see RecomputeChromatinVolumes_150330).
  • simple attempts at linear rescaling based on radius of gyration, computed volume, rg ratio, don’t suggest that such an approach is promising.
  • not sure the z-data is quantitatively accurate enough for these measurements.
  • blue data pretty reliably still for large regions in all dimensions, but for large black and large yellow there is a systematic depletion of the data at the z-extremes due to detection efficiency issues, which causes these trends to curve down. Images look okay but quantification not as good.
Posted in Summaries | Comments Off

Protected: MERFISH practice talk – Hao

This content is password protected. To view it please enter your password below:

Posted in Summaries | Comments Off

Thursday 03/26/15

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
    SingleEmbryoTimeSeq
    Hox Activation
    SingleEmbryoHoxAcitvation

Ordering control probes

  • submitted orders to Alec for BioSearch / Stellaris probes for Kr and tll as positive controls.
Posted in Summaries | Comments Off

Wednesday 04/25/15

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
    EmbryoSectionFPKM
Posted in Summaries | Comments Off

Tuesday 03/24/15

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
Posted in Summaries | Comments Off

Monday 03/23/15

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
Posted in Summaries | Comments Off

Tuesday 03/17/15

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…
Posted in Summaries | Comments Off

Monday 03/16/15

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

KcCNV

Exploring metrics

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

CVofLocalDensity

NormSurfaceAreaVsNormPerimeter

PerimeterToAreaRatio

medianFracLocInRg

asphericity

rgOverV

To do

  • CoV of pdist matrix
  • this looks promising:

histPdist

Posted in Summaries | Comments Off

Friday 03/13/15

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
Posted in Summaries | Comments Off