9:00 am

Job Apps

• working on figures for Research Statement (11a – 2p, 8p-10p)
• some progress on corresponding text.

Manuscript

• tweaking alternative version of EDF 3. (9:30 – 10:45am)
• revising response to reviewer letter 2pm – 4:30 pm, 6:00pm – 7:30pm

STORM hal updates

• brief tutorial from Hazen (11:15 – 11:30)
• see hb-storm-control (development branch) on STORM2
• new settings (examples in Hazen documents)
• camera has multiple feeds
  • settings file can specify feeds
  • these can display just a sub-field of view
  • these can also integrate a given number of frames
  • or just display every Nth frame
  • they can be told to save, they save with the name of the main file plus appending a user defined flag which you name the feed
  • camera1 (the whole field) is also saved by default unless you set save to False.
• steve settings are now all apart of hal settings
• no longer will have separate branches for separate setups, everything is in the parameters file
• Hal needs to be running first (or right click steve and select Query Hal) to get the objective and settings
• still need to add the 1.5x slider option to new hal parameters (from my Steve parameters).
• Hal/steve/dave all reset the python paths so you can have multiple functional copies of storm-control
• movie saving is now handled by main thread, not by camera. Keep an eye out for movies that drop frames or hang while saving (if camera drops frame hal might not know).

9:00 am – 7:00 pm, 9:00 pm – 10:00 pm

Manuscript revisions

• revising main text in response to XZ comments
• figure adjustments in response to XZ comments
• discussed main text revisions
  • simplified Ph KD discussion
  • simplified PRE discussion
  • simplified fixation controls and discussion
• further formatting of main text
• adjusted examples in fixation controls, show more detail.
• sent to collaborators

10:30 am – 7:00 pm

Job apps

• writing research statement

Figure revisions for chromatin

• see XZ notes

Experiments

• DNA digestion following IVT (dilute 2x add DNase I from NEB IVT quick kit)
• RNA purification
• quantify yield
• transfect new KC cells. (5:15 pm)

10:40 am – 1:30 pm

New Ph KD

• set up new Ph KD with last drops of previous dsRNA batch for ph-p and ph-d (well, I still have some ph-d left). Also did Pc KD as a bonus.
• ran 10% short on ph-p. And I’m not sure if this dsRNA is still up to snuff, its been thawed at least 5 times and in the -80 for over a month.
• knockdown started at 12:10 pm.
• started prepping new dsRNA for ph-p and ph-d.
  • PCR / cleanup column doesn’t seem to be ideal — I spec’d the DNA (~70ng/uL and 160 ng/uL respectively for ph-p and ph-d) and the 230 peak is as high as the 280.
  • set up T7 reactions anyway. Might run a gel after the DNase treatment and RNA cleanup (do tomorrow?)
• should probably do another round of QPCR on these RNA isolations before deep sequencing to check KD given issues.

Job app stuff

• working on revised statement.
• updated CV with CB comments.

9:05 am – 5:15 pm

Chromatin manuscript

• revising ED fig 3
  • I think we can make the fig 1 images work
  • might need to do denoising requested by reviewer
  • also tuned up previous examples and improved contrast (still only 1 example per each, was requested 3).
• finished revised letter fig 2 with 3 3×3 panels comparing centromeric, repressed and active chromatin
• added 300 cells into ED fig 4 and reformatted the fonts and labels
  • modified script to output “A-” and “I-” to meet the very latest of the naming conventions (didn’t propagate into allData structure yet).
• revised main text, discussed changes with BB. Sent to XZ
• XZ requests more deep sequencing.

9:15 am – 10:00 pm

Manuscript

• revising main text

chromatin experiments

• helping BB do deep-seq prep
• KapaQuant results:
  
• concentrations lower than expected, (only most concentrated of the dilutions look good)
• suspect a problem with the beads
• Tapestation quantification — no detectable library smear. Prep failed.

10:00 am

job app stuff

• working on description of institutions with due dates for letter writers

Career Advising appointment

• scheduled for Oct 16th, 10:00 am
• email cover-letter and CV to in advance kargere@fas.harvard.edu

Ph manuscript

• tweaks to fig 5
• discussed modeling.

3:45 pm – 7:45 pm

STORM

• C03 imaging finished ~4:00 pm (17 hours)
• replaced with fresh buffer, setting up new tile scan.
• Fixed Kc cells plated on 18mm round coverslips last night.
• started new imaging round
• started DaoSTORM running on Morgan to process last nights’ data
• set up Analyze on Arrival to run and process data currently being collected.
  • It generates mlist files but currently all are size 0, so I’m not sure it’s working.
  • maybe it’s just going really slow, I’ll check back later.

12:15 pm – 5:30 pm, 8:40 pm – 11:20 pm

Ph manuscript revisions

• went through and main text revisions, updated and replied
• went through supp text revisions, updated and replied
• updated Supp Figs (updated SF1, check with NF about SF11 and 12)
• went through response to reviewers, updated and replied
• sent all docs to XZ for comments
• approved. Sent all docs to NF.

Chromatin manuscript

Main text revisions

• start adding new sections in response to reviewers

More data!

• imaging more examples of C03 to get over 200 cells (on STORM2)
• switching pretty well, selected 57 regions, 60,000 frames, started imaging 11:15 pm

Starting on Figure changes for chromatin manuscript

• (noted below)

Figure changes to do

• Figure 1
  • Add Pc knockdown results to panels c and d (remove ED Fig d)
  • BB
• Figure 2, no change
• Figure 3
  • angle bar graph labels.
  • provide multiple examples for the new images (3?)
  • shrink panels in b to be same size as in Fig 2
  • Try to compress c and b and keep half page
  • STARTED. See draft on MORGAN as AI file (extra space for alternative images)
• Figure 4
  • remove panels a and d
  • compress g and h below the other panels
  • reduce figure to half column width
  • First pass done. Font sizes should drop dramatically to match typical nature — this can be reduced to 50% size probably without switching the font.
• ED Fig 1.
  • keep only images of cells fixed with our method (PFA)
  • Put all bar graphs on same plot
  • “standard” rendering of telomeres. These look like gaussian blurs, should be shaped blobs, should be able to see single molecule background.
  • provide multiple example images.
  • BB will do
• ED Fig 2: no change
• ED Fig 3:
  • switch a and b.
  • render b using examples from Fig 1.
  • contrast and color STORM images in b so they print as in Fig 1.
  • provide multiple examples of this panel to chose from.
• ED Fig 4: no change (unless it can split)
• ED Fig 5:
  • 3 panels only. relabel a, b, c (cut forth panel).
  • COMPLETED
• ED Fig 6:
  • added panels c and d belong in ED Fig 8 instead.
  • COMPLETED
• ED Fig 7:
  • delete this figure
  • (Will need to update numbers)
• ED Fig 8:
  • color controls, PcG targets genes, and Knockdown targets. Add separating lines. For both panels a and c.
  • panel b (RNAseq data) plot as bar-graph ratio as in a.
  • panel d gets cut (this data goes in Fig 1 c and d).
  • add panels c and d from Fig 6.
  • COMPLETED
• ED Fig 9
  • split off new panels as a new ED Fig
  • match sig figs above graphs with those in table.
• Letter Fig 1: Hi-C data
  • for Xiaowei, add all the Hi-C data comparisons shown previously
• Letter Fig 2:
  • keep current version just for Xiaowei’s reference
  • create new figure for the reviewer. Just images, no quantification.
  • 3 x 3 STORM panel of ~.5 Mb domains, ~100 kb domains, and 50 kb domains of Active, Repressed, and Centromeric.