Alistair's Lab Notebook

9:30 am – 7:30 pm, 10:00 pm – 12:30 am

Tasks

• Revise figure for XZ

New Library Design

• Final checks for lib5
• send Fasta file to Marcelo and request for quote to Alec and Matt

Sequential staining

• Lib2 F05 + F06.
• staining S1, 1:500 in hybe dilution buffer, 5:30pm
• accidently tripped over objective heater, came unplugged. Temp dropped to 28C.
• replugged (has 2 ports, use front port to go to heater). Heating back to 37C.
• 6:35pm finish Hybe, washing with 50% Formamide in 2x SSCT.
• Forgot to add beads. After wash (~6:50pm) remove sample, add beads, replace sample.
• Focus lock doesn’t work if stage is clipped into the bottom

Simulations

• tried ultra-stiff polymer (increased K by 1000)
• started saving template scripts in save folder so we can go back and trace the parameters.
• Installed dependent 32-bit python 2.6 packages for OpenMM on Monet (as not to interfere with existing 64 bit python 2.7 packages required for storm-analysis and storm-control projects.

10:00 am – 5:45 pm, 8:30 pm – 12:05 am

Lab meeting

• see notes on RC discussion
• see notes on conference update

Other meetings

• project 2 team meeting, 4pm
• chromatin project data update, 5pm
  • # send new plots to XZ tonight !
  • # send outline of figures to XZ: (1) schema. (2) regions have destinctive features with zoom ins. (3) scaling of area (4) internal domains (5) boundaries (6) model

Chromatin Project

Presentation Prep

• making slides for today’s meeting

Cell staining and imaging

• wash out excess probes D01 – D03 (stained yesterday)
• finish STORM imaging of sample L3B11.
• B11 also lost focus overnight (5am). Fortunately the findsum caught this error and stopped analysis before remaining sections were bleached.
• start imaging sample L3D01

Analysis

• Analyze B09 data through image 21 (53 spots), have 34 images
• Processing B10, B11, F01 and E12 data. E12 mislabeled as E11 in dax files. Running on Tuck and Cajal
• Analyzed F04F02 (black left + En) and F04 + F03 (black left + E(Pc)).
  • localization count pretty low.
  • 750 channel often quite weak.

Simulations to do:

• simulate random walk polymer, then simulate exponentially distributed counts per spot. See what the effect is on radius of gyration.

4:00 pm – 11:00 pm

Lib5 assembly continues

• found bug in probe sizes, new 40mers had been overwriting some of the 30mer sublibraries.

Data Analysis

• re-analyze B02 (+ more cells)
• finish analysis of B06
• analyze more cells from B05
• analyzing B07 and B08

STORM

• imaging B10 sample

4:00 pm – 7:00 pm

(return from Maine).

Goals

• Finish construction of Lib5 (with chromatin lib 4).

Library Design

• Running CompileOligoArray Script on Library5 40mers.
• didn’t screen out repeat probes and rRNA blast out of ProbeData structure in previous runs. Now correcting this.
• Problem: how to remove individual probes from genes that have off target hits with a single BLAST (obviously I can BLAST all genes indvidiually, but I think this is going to be very slow). BLASTing one big list doesn’t take much time.

9:30a

Lib5 prep

• need to split up black regions
• spent several hours needlessly parsing lib3 to encoproate into lib5.
• building stringent BLAST against highly expressed sequences and ribosome sequences.

Discussions

• discussing with Junjie methods for statistical testing of data, methods of cross-talk subtraction, and methods of data normalization
• Cut ~1000+ probes (~2% of probes) based on this final BLAST.

Reconfiguring Mosaic Viewer for Guiping

files, locations and descriptions

mosaic_to_matlab.py (now a part of storm-control/steve)
stv2mat.m — just calls the python script mosaic_to_matlab.py. Latest example saved in Chromatin/Scripts
viewSteveMosaic.m — function written to render the new .stv / .mat formatted steve files. part of matlab-storm\GUIs\Library\STORMrender\.