Alistair's Lab Notebook

10:00 am – 8:00pm

10 am – 11 am: Ph simulations

• migrating Ph simulations onto Morgan.
• running Ph simulations
• maybe much shorter equilibration time and just many more rounds of simulation would be better
• running lots of short simulations across a broader range of Ph. This round wont finish before tomorrow.

11:00 am – 12:45 am

• revising MERFISH proofs

MERFISH meeting and probe design 1pm – 5pm

• 20mers 66C – 76C not enough

OligoArray batch launch issues

• read-write speed of fasta files seems to be limiting new launch — 30 jobs quickly launched but none complete
• troubleshooting OligoArray speed
• at crosshybeT of 99, still get lots of ‘nonspecific oligo ignored at this time. Later get
• launched a 40 to 50% GC content version of scan (this is going to get less than 1000 genes) [no other stringency parameters]
• launched a 15 to 75% GC content (this isn’t much of a cut).
• stopped previous 20 mer building at 35869 of 37442 probes. (lib11 prep folder: 15-04-03_Probes_20mers_t66Cx99Cs99C).

Probe numbers

• Previous 30mers, FPKM > 0.1 and >100 probes = 2197
• New 20mers, FPKM > 0.1 and >100 probes = 3929
• Previous 30mers, FPKM > 1 and >100 probes = 1856
• New 20mers, FPKM > 1 and >100 probes = 3026
• New 20mers, FPKM > 1 and >50 probes = 6920 (4 2ndaries per probe we still have 50 per hybe).
• New 20mers cuts: Tm 66 to 76.
• Of 37608 genes in our database, 12,016 are longer than 2 kb (i.e. 100 probes if we just uniformly tile with 20mers). Not sure why I am losing so many…

5pm – 8pm: more Ph simulations

• need to explain limited binding sites (K27 model) and its effects on cluster size distribution.
• I think actually binding is too strong, data suggests there are fewer

9:00 am – 11:45 pm

Tasks

• revise Text for chromatin paper
• Extended data figures for chromatin entanglement (done)
• revise Fig 4
• Histone images for Doory (sent)
• Revised model figure for Ajaz
• write abstract for ASBMB on MERFISH (see post)
• write abstract for Soc Dev Bio on chromatin

9am – 2pm

• revising chromatin text
• polishing abstract with Bogdan, 10:30am – 11:30am

5pm – 9pm

• entanglement supp figures
• revising figure 4
  • update colors, labels, graph sizes
  • add images of subdomains
  • cleaned up model fig code
• rewrote caption
• updated into text, now version 4-4.

9pm – 11:45 pm

• waiting for BB to read MS
• should we change to all volume no Rg?
• generated semilog-y plots for internal scaling data and linear plots. All in dropbox\Figures\Version7\Figure2
• sent to XZ

Data Analysis

• running E04 E05 data through Chromatin Cropper
• errors in automatic load after splitdax. needs troubleshooting

Potential conferences

• http://www.cell-symposia-stem-cell-epigenetics.com/
  • Ren, Zhang, Wysocka
• Keystone symposia (but nothing looks relevant. This would have been good: http://www.keystonesymposia.org/15Z2).

10:10 am – 5:00 pm, 7:30 pm – 11:00 pm

Discussion notes

• Lab meeting (see notes)
• discussion with Bogdan on research projects
  • anticipating review requests
  • things to strengthen the paper appeal
• revising manuscript

Computer troubleshooting

• reboot seems to have fixed oligoArray. Now running (slowly)
• deleted and remade account for JM to fix corrupted user accounts

Manuscript Revisions.

10:00 am – 12:15 am

Chromatin manuscript

• revising abstract and intro as discussed
• updated figures. I don’t think I like the new color scheme.
• made changes to main text to match figure changes
• sent latest draft to JM and BB for comments, 11pm

Oligo array issues:

• Morgan doesn’t have java
• installed java. restarted matlab. now java runs
• java can’t find blastall –> invalid complaint about database. Not sure why it can’t find LegacyBlast
• need to add LegacyBlast and OligoArray aux to file paths in Environmental Variables (Advanced System Settings) I guess, how did these get wiped?
• rebooted MORGAN (porbably not necessary, see if all my files get wiped now).
• Yup, ‘you’ve been signed in with a temporary profile. You can’t view your files, and files you create in this profile will be deleted when you sign out’ — all after a long sign in delay.
• tried creating new AlistairTemp account from Administrator Account. See if this one does not get corrupted.

9:00 am – 1:00 am

Chromatin Manuscript

• working on revisions
• discussion with XZ, BB, and JM on organization

Recommended changes

Intro

• first paragraph on structure
• second paragraph on chromatin state

Summary paragraph

• organization at different scales.
• ~one sentence referring to spatial organization with references
• ~one sentence referring to organization by state / chromatin type
• ~Not known: whether states differ in physical organization.

Other changes

• figures: see notes.

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.