Alistair's Lab Notebook

11:00 am – 1:15 am

PcG RNAi

New Knockdowns and cell culture

• plate Ph, Pc, and mock RNAi cells onto coverglass
  • density looks okay modulo a decent number aren’t sticking
• isolated cells for RNA extraction
• fixed plated cells to coverglass for imaging
• Psc null cells
  • starting to dye for not changing media (despite growing too slowly to be confluent).
  • replaced media on small plate and scraped plate (both these look pretty decent toward confluency).
  • combined media into new small vial, maybe detached cells will attach and settle (took > 24 hrs to attach last time)
  • made new Psc media (using modified Schneider’s from Lonzo and HI FBS).

Imaging BX-C in Ph-KD

• finish imaging of L2F03F04
• start imaging of L4E24

Analysis of BX-C internal domains in Ph-KD

• still fiddling with export options for ChromatinCropper2
• working on building ChromatinCropper2 GUI. This always takes longer than I think.
  • meanwhile I’m working out what I hope is a better general system for GUIs.

Cell cycle studies

• prep cells for hybe
• hybe BX-C to cells.

10:00 am – 11:00 pm

To do

• PRE correlation analysis
• expression correlation analysis
• design and Green domain probes
• finish updates to ChromatinCropper2
• analyze internal domain data

Correlation analyses

• repeated PRE analysis as discussed:
  • recomputed using Cavalli/Tanay labs latest PRE predictions
  • examined distance from trend as function of PRE density
  • explored PRE density and number vs. volume (compared to length vs. volume)
  • could trivially extend to Rg
• loaded expression data, see some weak correlation to explain outliers
• also see (surprisingly) correlation with CTCF peak density to explain outliers
• should combine these two.

STORM analysis

bead calibration

• chromatic calibration still works okay with defaults
• z-calibration: would like to rewrite this function. Less automatic selection of curves and more user input.

Live imaging

• attempt repeat of live imaging
• 2nd round looks good.
• repeated issues with sample movement after pipetting
• really should remember double-stick tape next time.

9:00 am – 12:00 am

STORM live imaging

• construct doesn’t show expected pattern
• fixed issue with 561 power (half-wave-plate was turned) so the beam entered the polarizing beam-splitter the wrong way.

STORM Ph-KD imaging

• imaging Ph-KD L2F04 sample
• seems to switching well.
• seems to be saving well.
• need to take 3D calibration movies in the morning.

MERFISH analysis

• see team note-book posts on 2 color L12 data set

Chromatin Data processing

• started running PH-KD L2F03 data through DaoSTORM
• also running bead data through fitting

Ph project

• revised replies to reviewers
• sent draft version to XZ

9:45 am – 11:15 pm

Batch data analysis of confocal live imaging of fixation effects

Matched feature aligned images:

Positive Control:

A few more examples of positive control

Signed up for Elyra training

MERFISH

• analyzing L12 data
• see notebook post

STORM

• imaging session start delayed to 6:30 pm (from 5:00 pm).
• attempting STORM imaging of L2F04 Ph-KD on STORM2
• focus lock doesn’t work (defocuses)
• 561 laser intensity very low (turned out to be the half-wave-plate polarized the beam in the wrong direction)
• switched to STORM3
  • quadview is badly out of alignment (apparently due to mis-treatment of the unit?)
  • beam behaves as if there’s a fun-mirror/lens in the path causing very weird distortions to pointspread function as spots move in and out of focus.
• aborted attempt to image tonight (~11:15 pm).

9:15 am – 5:05 pm,

Goals

chromatin STORM

• finish STORM session of L2F03 dat in Ph-KD condition
• take bead movies, transfer data
• start fitting movies after transfer completes
• setup auto-transfer (?) for next time

Ph project

• send Ph data on downsampling for Fig 1 graphs to NF an AW
• analyze effects of down-sampling on fig 2 results

STORM2

• new pixel size for 1.5x slider and 60x objective = 0.192 um (or 0.195 um)

Chromatin Project To Do list

• live imaging
  • analyze new live imaging data from confocal
  • PCP imaging of telomeres (in U2OS?)
• RNAi experiments
  • set up new RNAi for Pc(v2) and Ph-p + Ph-d
  • prep for sequencing Ph KD cells and WT cells.
  • repeat qPCR experiments for Ph KD — need more robust replicates
• centromeric chromatin imaging
  • Design library for green regions
  • assemble and process existing data on green regions
  • image AATAT centromeric region in Kc (should be good live imaging target too)
• PRE analysis
  • re-analyze PRE-density as a function of volume using new PREs, see if its a primary effect.
  • also compare difference from trend line with PRE density, see if its a secondary effect.
• Hi-C analysis
• Active chromatin analysis
  • correlate TSS density and difference from trend line
  • correlate expression level and difference from trend line
  • correlate insulator protein density and difference from trend line

Chromatin Project work

• ordered CycB antibody
• analyzing mmaple3-dCas9 data on Morgan
  • no obvious signs of telomere labeling
  • not a sharply defined nucleolous border
    
• working on analyzing confocal live imaging data: see post

11:00 am – 3:30 pm, 6:30 pm – 11:15 pm

STORM

• STORM2 froze during the night (click error provided an out of frame crash in python — built in check doesn’t catch it.
• debugged this with Bogdan, changed catch statement to check size of the object queried, not just the expected size of the image.
• restarted imaging of Ph-KD L2F03 with fresh buffer
• acquisition still looks good at movie 18. And rolling.
• need to take z-beads and chromatin beads tomorrow before Colenso starts.

Planning

• need to design and order new Green probes.
• need to analyze existing green data.

MERFISH

• figuring out parsing of new data schema
• trying to process L12 two-color data for analysis with Pipeline 5 or updated pipeline 1.

Nuclei live imaging

• took time lapse images of Hoechst stained nulcei (2 year+ expired DRAQ5 didn’t look that good at 1:10,000)
• took images of mock fix (replaced growth media with more growth media)
• took images of formaldhyde fixed cells
• took images of MeOH fixed cells (clearly shrunk)
• need to do some coding to analyze these.
  • probably worth some manual playing with the Fiji tools too.
• some issues with Z-focus and x-y registration, largely sorted though.

Ph-Project

• check that numbers don’t change much upon down-sampling
  

Review

• reading article, drafting comments of first impressions
• very long methods section, reading imaging methods in detail

9:00 am – 5:00 pm

Lab meeting

• see notes

Discussion

• talk with Ting
• talk with Xiaowei

chromatin project

• project update meeting
• see google-docs slides

Ph Project

• need to downsample localizations (2-fold?) an re-compute statistics and show similar distribution.

11:00 am – 7:30 pm, 9:30 pm – 10:45 pm

(9:00 am – 11:00 am volleyball practice)

Chromatin

Cell

• plated Kc167 cells (in SFX) on glass
• my more recent flask with the short Trypsin treatment is doing well.
• the original passage with the longer Trypsin exposure is not looking so good.
• tried removing Psc- cells by blowing (without Trypsin). works a little bit, not at all like with Trypsin.
  • added a little scraping an moved cells to new vial
• tried imaging cells live labeled with Hoechst – doesn’t bleach much or blink
• tried imaging cells live labeled with Draq5 – doesn’t bleach at all (or switch to dark state to blink)
  • tried both culture media and STORM BME imaging buffer.
• Invitrogen has a number of other cell-permiable dyes: https://www.lifetechnologies.com/us/en/home/references/molecular-probes-the-handbook/probes-for-organelles/nuclear-and-chromosome-counterstaining-and-nissl-stains.html#head1

Cell cycle markers

• CycA (DSHB)
• phospho-H3 (M-phase specific) see here (fig 1, red)

Culturing Psc- cells

• tried passage by scrapping — disaester, cells die
• tried trypsin first spin looks good. No cells came down in PBS spin (sparse cells). Don’t look good on plates. I think I killed these.