Alistair's Lab Notebook

10:30a – 11:45p

Data analysis

• Launch STORM analysis for bead data
• crazy transpose now between conventional images and STORM images in F02F03 data. May affect all recent data?
  

To do:

• Finish fellowship progress report
• 2 color imaging of other en-locus boundary regions
• draft fig 3 for project 2 to show XZ + meeting
• analysis of 2 color
• fix new cells for new multi-color stains and continuing analysis of more yellow regions

STORM imaging

• multi-color imaging F04F02
  • cell 0001: 1 uL pure COT + 2.5 uL MEA. Not too bright.
  • cell 0002-0003: brand new glox. 10 uL DMSO dissolved COT. 2.5 uL MEA
    • beads clearly bleaching fast.
    • domains look spread out / way too big in 647.
• single-color imaging O/N L3B04

Cell culture

• cells a bit over-grown, not passaged for nearly a week.
• passage cells
• plate new cells for fixation
• original plating density a bit to high. removed excess cells and added new media.

Modeling

• OpenMM tutorial with Bogdan
• implemented spherical confinement potential. See tuck c:\polymer\ for code.

New stains

• F06p1-F05p3, F03p1-F04-p3, no secondary.
• B05-p1 + .5ul A647
• plate 40 mm cells

10:00a – 10:00p

Polymer sims

• periodic loops and sticky

• show more positive slope than just perodic loop model did (substantially).

• equilibrate slower.

observations for yellow

• small 10-35 kb regions seem to be larger than expected by scaling extrapolated back from the bigger regions — maybe there is a typical contact length here?
• simulate typical scale contact (fixed pairwise contacts, average of 10 nodes.

11:00a – 11:30p

Lab Volleyball, 9a-11a

Meetings

Project 2 weekly meeting, 12p-1:30p

Meeting with Ting, 2p-4:30p

• Kc167 cells mostly tetraploid for all chromosomes (II, III and X). May vary in chrII but do not appear to in recent experiments.
• ~78% paired (see historic Wu lab publications). Consistent with my estimates of ~80% paired.

Meeting with Bogdan

• working on Drift Correction with blead-through beads / integration into ChromatinCropper

STORM

• finish staining / washing samples B02-B04
• imaging B02

Coding

• running simulation on blue domain loops

Housekeeping

• change media for cells.

10:00a – 10:50p

Goals

• team meeting — need replies for XZ and Tani
• Stain new yellow domains
• troubleshooting multi-color

Chromatin Analysis

• preliminary analysis of radius of gyration.
• wrote code to compute flists (now a Chromatin Function) for old data which just had parameters vlists, and final pixelated images. This should be useful for matching the resolution
• a few outliers
  • G06 still (I think this one needs to get redone because I believe it missed the RNase treatment)
  • C02 is also substantially off on radius of gyration, though not my prior area calculation so much. This bears looking into

STORM

• Playing with multi-color analysis of F03-F04
  • round 1: day old TCEP buffer
  • round 2: new TCEP buffer
  • round 3: new COT MEA buffer, low MEA
• Try COT + TCEP? (TCEP pretty annoying in not being able to see conventional spots. With the background issues I think this is going to be a disaster).
• Try more or fresher COT? Fresher MEA? Epoxy sealed chambers?

Simulations to do:

• polymer chain interactions — entangled chains with separated centroids. unentangled chains with non-separated centroids.
• A few regularly spaced strong contacts in an otherwise weakly sticky polymer (can’t be too sticky, or it will just collapse to the space-filling case always). Simulating this now.

10:00am – 5:45pm

Hal froze last night on movie 4! :(. Restarted hal, restarted imaging.
Really should remember to check this before I leave — this must have happened before I went home last night near ~1 am, since the scope was running by 7 pm or so.

Windows on Monet froze again — windows explorer sucks all the memory (32GB) and freezes while browsing my lab notebook images 2014 folder. Incompetent operating system.
Dell Aurora keeps forgetting the correct boot disk upon restart for security updates. This is annoying. Cold shut-down reboot and it loads the correct boot disk. Clearly another bug.

Goals

• work on lab meeting presentation
• start work on fellowship Progress Report for Damon Runyon (due at end of month)
• launch analysis of unprocessed STORM data

STORM analysis (ChromatinCropper)

• Analyzing E04 data. Very high background.
• Analyzing E04 E05 chrX:1907754-2047754
• Analyzed E06, E07, E09, E10

STORM

• restarted imaging F03F02
• set up new imaging of E01 (repeat, now doing 175K frames instead of 65K).

8:45a – 12:45a

9:00a-11:15a, lab volleyball practice

Probe synthesis

• B02-B09 3ul P1-A405, 10 uL RNA,
  • plus 5 uL RT buffer, 2 uL dNTP, 1.5 uL ddH2O, 1.5 uL inhibitor, .2 ul RT
  • Master = 27 uL P1-A405 + 45 uL RT buffer + 18 uL dNTP + 13.5 ddH2O + 13.5 uL inhibitor + 2 uL RT
• B02-B09 3uL P1-A647, 10 uL RNA,
  • 5 uL RT buffer, 2 uL dNTP, 1.5 uL ddH2O, 1.5 uL inhibitor, .2 ul RT
  • Master = 27 uL P1-A647 + 45 uL RT buffer + 18 uL dNTP + 13.5 ddH2O + 13.5 uL inhibitor + 2 uL RT
• F03-F11 4uL P1-A405 15 uL RNA,
  • plus 6 uL RT buffer, 3 uL dNTP, 2 uL inhibitor, .2 uL RT
  • Master = 36 uL P1-A405 + 54 uL RT buffer + 27 uL dNTP + 18 uL RNAsin + 3 uL RT
• F03-F11 4uL P3-A405 15 uL RNA,
  • plus 6 uL RT buffer, 3 uL dNTP, 2 uL inhibitor, .2 uL RT
  • Master = 36 uL P3-A405 + 54 uL RT buffer + 27 uL dNTP + 18 uL RNAsin + 3 uL RT

Ran oligo cleanup on all 32 new probes. Still need to spec and label these probes. Currently brief labels are on the sides of the tubes and all tubes are in a new cardboard box in my -20C.

STORM

• imaging F03-6 and F02-7 double stains

10:00a – 11:30p

Project 2

• see notes

Chromatin Project

New Probes making

• L3: B02 – B09
• L2: F03, F04, F05, F06, F07. L3: F09, F10, F11 (all 2 color),
• running QPCR (try 2)
• over-ran a little bit, amplification happens quite rapidly (less than 8 minutes) between cycles 25 and 30.
• PCR cleanup of all samples
• Setup T7 IVT reactions, B02-B09 20 uL reactions (renumbered as 1-8). F03-F11 (renumbered as 9-16) as 40 uL reactions (want more RNA to do more RT reactions)

Bogdan and black chromatin

STORM

• Finished imaging L3F04-p1-A647 + L3F03-p3-A750
• Start imaging L3F02-p1-A647 + L3F03-p3-A750

Chromatin Analysis

• E01 and E02 samples seriously under-stained / under-sampled / too-short images. Apparent domains are large, but sampling density is insufficient to resolve details and keep domain connected. less than 1000 localization for many of these 250 and 175 kb (respectively) regions.
• Analyzed E06
• Analyzed E07
• Analyzed E10 up to image 25 (50 spots)

matlab-storm

• still trouble-shooting
• fixed some bugs in parameter passing from GUI-popup window to main analysis function
• Warps do not seem to be getting applied to data, points aren’t moving.
• My ‘translation rotation’ function for ‘WarpPoints’ seems to be in error.

9:30a – 5:00p, 10:30p – 12:00a

Goals

• Finish stains
• 2-clr STORM imaging En TAD regions
• Group meeting prep.
• Communication
  • email Graham
  • email Steph dates

Lab Meeting

• (laptop battery died, no e-notes).
• 750 with adaptive optics?

Probe making

• RT-PCRs didn’t work. (maybe screwed up primers)
• Repeat with B01-B08. These did work. Started amplifying around cycle 22. See PCR curves. 45 cycles all saturated before cycle 40.

STORM

• imaged bead fields
• imaging F03-p1 F04-p3 lib 3 double color with MEA. Looks pretty good, plenty of switching.