Alistair's Lab Notebook

Manuscript

• paper revisions
  • finalizing response to reviewers with BB comments
  • discuss updated comments with XZ

Figure changes to do

• Figure 1
  • Add Pc knockdown results to panels c and d (remove ED Fig d)
• 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
• Figure 4
  • remove panels a and d
  • compress g and h below the other panels
  • reduce figure to half column width
• 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.
• 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).
• ED Fig 6:
  • added panels c and d belong in ED Fig 8 instead.
• ED Fig 7:
  • delete this figure
• 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.
• 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.

Ph project paper

9:30 am – 7:00 pm

Finalizing draft of response to reviewers

• polishing figures a bit (removing boxes, fixing axes etc)
• finish writing missing captions
• 9:30 am – 10:30 am

communication

• catching up on communication emails (10:30 – 11:15)
• coffee with NB
• reply to pairing hoedown invitation
• reply to meeting planning for CONTI retreat

Discussing deconvolution

• doesn’t shrink area estimated much
  
• maybe this is a problem? More iterations than the default do further tighten the PSF if you apply it to itself. In 10 it doesn’t shrink down to a point but it does in 60. Lots of iterations on sample data does not reduce it to a point, however the background noise goes a bit crazy.

QPCR

• 3 Ph-KD with corresponding mocks (samples 1-6): test with 2 alternate ph-d primers (12 wells)
• 1 Pc-KD with corresponding mocks (samples 6 & 7): test with 2 Pc primers (4 wells)
• I had additional Pc-RNAi data from 7/3 with 2 replicates. Maybe I can find these samples again.
• sample order:
  • row 1, col 1-6 ph-d primer set 1 (cDNA samples 1-6 from 8-14: ‘Ph 7-7′,’mock 7-7′,’Ph 7-14′,’mock 7-14′,’Ph 8-8′,’mock 8-8’)
  • row 2, col 1-6 ph-d primer set 2 (cDNA samples 1-6 from 8-14: ‘Ph 7-7′,’mock 7-7′,’Ph 7-14′,’mock 7-14′,’Ph 8-8′,’mock 8-8’)
  • row 7, 8 Pc primers 0 and 5 in Pc 8-8 KD and mock 8-8 (described in matlab script qPCR_RNAi_PcGs_150910b)l
• data looks good, Ph-d primer 1 didn’t work but primer pair 2 looks decent.
  
  

RT for QPCR from Pc RNA

• samples 7-26 mock, 7-26 Pc v1, 7-26 Pc v2, 8/8? Pc KD (v2)
• RNAse H treated (ran a bit long, 50 min at 37C instead of 20 min)
• Ran DNA cleanup column, eluted in 30 uL
• diluted 20 uL into 40 uL 10 mM Tris pH 8.0
• setup new QPCR
  • 4 cDNA samples, 11 primer pairs.
• 50x master mix: 625 uL 2x Phusion master + 312.5 ddH2O + 62.5 EvaGreen
• • 3 uL cDNA + 2.5 uL 50 uM primer mix
• QPCR plate layout
  • col primers: ‘Act5c’,’alpha-tub’,’gapdh1′,’Ubx’,’Abd-B’,’Dfd’,’Antp’,’en’,’Pc0′,’Pc1′,’Pc2′
  • row cDNA ‘Pc KD 8/8’, ‘Pc v2 7/26′,’Pc v1 7/26′,’mock 7/26’

9:15 am – 5:10 pm, 8:00 pm – 11:00 pm

Response to review letter

comparisons with conventional imaging

• working on deconvolution
• went back to fully automated quantification of conventional images
  • using half max to address thresholding — works pretty well.
  • some conventional images weren’t taken at very good exposure and should be excluded.
• selected 5 domains of each type with good conventional images that evenly span the scales
• created new images
• selecting conventional images to compare with STORM images at equal resolution (supplement)

Revising text

• addition captions

9:00 am – 8:00 pm

Revising manuscript

• New versions of all the main figures and all the extended data figures now started.
• Figure items: Still need to do
  • deconvolution of conventional images
  • add anti-aliased full size conventional images
  • analysis of Pc KD domains, add to Ph-figure
• Text things to do
  • revise captions for all figures
  • revise letter, match all figure panel labels
  • add images of figures into letter
  • add methods section for PcG KD and quantification
  • add methods section for NextGen sequencing

Prep

• wrote new script for designing primers and ensuring different regions are chosen (built on primer3 and NCBI live sequence retrival).
  • script is in ForReviewers folder of chromatin git project
  • saves images
    
  • green is QPCR primers, red is masked RNAi region. units are in bp
• ordered new ph-d primers for ‘same day delivery’ (ETA Thursday, how is that same-day, it’s Tuesday still. And these cost 13 each + 20 shipping instead of 3 and free shipping…)
• ordered more backup ph-d, ph-p and Pc primers

Communication

• NF inquire about paper
• discuss recent Fraser Luscombe and Elderkin paper.
• XZ will get replies back this week.

10:15 am – 8:40 pm, 9:40 pm – 12:00 am

tasks

• discuss quantification approaches for live data
• working on figures
• practice talk for CONTI retreat/review
• working on figures (see git and figure notes)

CONTI grant evaluation

meeting organization

• first: Tako Hensch, microRNA — AGO2 is imprinted
• Lichtman then Dulac then us?

Bogdan Talk: Chromatin structure

• ‘identical’ probably too strong for imprinted genes
• one allele is 1.5-2x larger than the other in imprinted tissue
• look at this ratio for non-imprinted genes (or same genes in non-imprinted tissue).
• whose maternal whose paternal?
• imprinting is highly tissue specific

Feedback

• too long (maybe 30 min?)
• intro is good (CD). why is structure important at length scale of genes (interactions matter?)
• cut the internal organization scaling laws.
• quantification of multiple domains for interaction (within and between domains).
• intermixing between domains needs a contrast.
• XZ likes the schema of why dense chromatin is harder to transcribe.
• conformation changes, cell-specific differences (single cell vs bulk).
  • kidney vs. brain validation (not)
  • show well studied example for which there is data (bulk).
  • large scale, single cell, 3D study. — imprinting in brain is 40:60 – is this per cell or only in average?
• IGF H19 data
  • preliminary but intriguing difference between allele size.
  • mark what’s labeled (full cluster of H2 IGF)
  • link spidery domain to active (just say provocative similarity)
  • don’t mention loops

MERFISH

• feedback to follow
• use Allen Brain atlas to motivate one gene at a time. No correlation analysis
• emphasize single cell resolution, high detection efficiency.
• much too long.
• less technical validation, more emphasis of the scale.

DJ

• behaviors in general
• instinctive behaviors — genetically programmed?
• Genes known for behavior
• The Medial Preoptic area of the hyothalamus: Distinct neurons active in the MPOA in different behaviors (Male infanticide, male parenting, male agreesion, male mating, female mating, female parenting)
• many definitions of cell types: propose that many of these are governed by combinatorial code of genes.
• RNAsin added to tissue to co-stain with antibodies.
  • differences in stains not so evident.

9:00 am – 8:00 pm

Presentations

• rehearsing lab meeting presentation (9am-9:30am)
• start cufflinks running on newly aligned NextSeq data (9:30 am – 10:00 am)
• lab meeting, 10am – 12:30 pm
• playing with deep seq data (needed to rerun cufflinks, overwrote files the first time since cufflinks default output is just genes.fpkm_tracking and I don’t know how to change it).
• grant meeting presentation rehearsals (2:30 pm – 5:00 pm)
  • (see notes)
  • additional notes for slide revisions on slides
• paper update meeting (5:30 pm – 6:30 pm)
  • time to start writing and making final figures
  • planning out plan of attack with BB

9:00 am – 7:10 pm, 9:00 pm – 10:00 pm,

Goals

• finish lab meeting presentation for tomorrow
• finish CONTE center presentation for tomorrow
• finish update slides for discussion tomorrow

Analysis

• analyze WT ANTC-G6 data

Morning tasks

• check STORM imaging
  • movies still going well
  • issues with beads in this sample, 561 is suddenly backgroundy but not a lot of beads in the focal plane.
• Auto-analyze on arrival
  • didn’t work. Changed defaults in RunDotFinder, seems to be working now.
• communication with CW, SN and DD about oligos and Biosearch/Stellaris

Analyzing Ph data

• cleaning up data
  • converting BB’s molecule lists back to standard molecule lists
  • need to convert xc and yc back to pixels
  • need to convert molecule lists back to structures of cell arrays rather than large structures
  • need to compute imborders
  • BB flists keep crashing my 3D plotter — haven’t converted everything very well yet.

File Organization

• Old TSTORM folder on my embedded DATA drive (D) is taking up too much space — consoldiating this on to the TSTORM data drive
• need to remember to delete after it finishes copying the ~600,000 tiny files over.

Imaging Ph in Ph-KD

• some difference in overall brightness — hard to quantify nicely since there is clearly background autofluorescence that needs to be accounted for.

RNAi NextSeq data

• data arrived today.
  
• Running Bowtie
  • error 1: mapped to two-stranded genome, all reads map multiple times
  • error 2: mapped to positive strand genome, 80% unique reads, 20% multiple alignment. But names need to match GTF file
• need to match expected folder structure — added these notes to my pipeline script
• would be nice to add cufflinks into this pipeline.
• need Bowtie Genome fasta to match reference names in genome GTF file given to cufflinks
• SAM file needs to be sorted — converting to BAM and sorting using sam tools
  • installed same tools for windows from here

Analyzing PhKD data

• serious lab issue on Monet. Going for the reboot. Hoping the Windows 10 install plan doesn’t kill it again.

10:00 am – 8:00 pm, 9:00 pm – 11:20 pm

Ph KD BXC-F6 further imaging

STORM

• sample chamber contaminated by Draq5 — can’t see dye switching

CycA

• cytoplasmic antibody stain still present after hybridization
  

Sample prep

• preparing new sample with BXC-P1 S1 and F6 in P3-750
• hybridized and staining, ~10:30 am

Ph antibody imaging

• rinsing out O/N primary stain.
• brief blocking
• incubate in secondary antibody (rb-488).
• some nuclei actually show good staining (some don’t). Maybe 1%-tween with block wasn’t sufficient permiabilization.
  
  
• repeat with Triton-X 10 min treatment, running Ph-WT and Ph-KD in perfect parallel

QPCR

• 9 primer-pairs + 8 samples
• 12.5 uL 2x Master, 6.25 uL ddH2O, 1.25 uL EvaGreen, 2.5 uL primer mix, 2.5 uL DNA
• 1 mL master, 500 uL ddH2O, 100 uL Eva Green (500 + 250 + 50);
• primers and cDNAs as labeled here:
  

Presentations

• working on lab meeting presentation
• working on figures for review to discuss at XZ meeting