Wednesday 08/20/14

9:30 am – 9:15 pm

Writing

  • working on / revising BW proposal
  • two more rounds of comments from Carl
  • comments from Jeff.

STORM Analysis

Serious problems introduced by microscope A

  • recent bead data sets collected on new version of software are NOT renormalized to maintain full dynamic range and constant contrast! Need to fix this in imagewriters.py
  • Dave destroys my conv_bk backup movies, does crazy unexpected stuff with power files and laser levels (blasts my sample with UV). This unanticipated behavior is highly destructive to my sample and data quality.
  • Unexpected behavior in dave is due to the entirely different way dave parses shutter files. This needs to be fixed before imaging more.
  • Running chromatic bead analysis. Quad view horribly misaligned — require match radius of greater than 30 pixels to match beads.

Literature

  • interesting article on transcription vs translation as determinant of protein levels. Also a good example of reusing public data sets.

Cover image

cover.tif

(Good article by Thomas, Hernan and Shawn as well).

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Tuesday 08/19/14

10:00 am – 11:00 pm

Writing

  • final comments back and forth on manuscript revisions and figures for Ph project
  • Ph paper sent to supporting middle authors for comments and revisions
  • working on BW proposal
  • got comments back from Carl on BW proposal.
  • Rewriting proposal from scratch, trying to improve clarity focus and impact.
  • completed first pass through new draft. Needs read-through and conclusion.
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Monday 08/19/14

10:00 am – 10:00 pm

Writing

  • revising manuscript and supplemental material for Ph Project
  • working on BW application

STORM

  • imaging L4E17. 2:00pm-10:00pm
  • imaging L4E17to19 (10:00pm – 10:00am)
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Sunday 08/17/14

10:15 am — 11:15 pm

Paperwork

  • working on essay for Burroughs Wellcome application.

Chromatin Imaging

  • Testing IR shutter for 750 imaging
    • wrote new communications/connections file for Toptica laser, saved to desktop for quick use Toptica750.ht (previous file copied from old storm2 computer doesn’t work — com port changed).
  • taking bead data
  • setting up to image new samples: L4E02-647 + E01-750
    • 647 worked much better this time — bright clear spots obvious in every cell.
    • 750 spots look pretty dim
    • originally made with pure BME buffer, spots were dim. Remade with MEA buffer, maybe a touch brighter but not strongly so. Remade with brand new MEA buffer, still not a dramatic change to 750 brightness.
    • original region selected went outside buffer resevoir over edge of PDMS. spots equally bright here (they were still bathed in buffer) but switching is poor (no buffer turn-over).

Python

  • working to get KnotAnalysis running on Odyssey (requires linux)
  • see emails from today’s date from RC computing with directions for running anaconda python and installing local python packages
  • Adding my directories to systems path once in python: import sys, then sys.path.append("/n/home05/boettiger/OpenMM/openmmPolymer")
  • KnotAnalysis seems to execute fine, though the number doesn’t quite make sense (200 monomers “simplified” down to 32 but with a knot number (crossing number) of 427.3 something like that. Why isn’t this an integer and how can it be substantially larger than the number of simplified monomers (or total monomers?)
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Saturday 08/16/14

5:30 pm – 12:10 am

Data analysis

  • Analyzing recent multi-color chromatin data with ChromatinCropper.fig
  • not dramatically different overlap/exclusion between yellow-yellow and yellow-black
  • yellow-black is however a ‘closer’ boundary (at least in the black-to-yellow direction) since the black domain is more compact
  • should also do some modeling on this to better inform intuition.

Some snapshots from today’s analysis

L2F6F7_example

L2F5F6_example

Literature

  • Interesting review article by Pirrotta on new papers arguing PRC1 then PRC2

Maintaining the living things

  • passage cells
  • flip fly stocks
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Friday 08/15/14

10:00 am – 11:30 pm

STORM

  • imaging failed last night
    • should have taken more alarm when the test image didn’t save
    • parameters were default to .647 format which didn’t exist, resulting in save error.
    • computer auto-rebooted last night anyway
  • Updating software
    • updated imagewriters branched off new / stable storm2. Now have .647 and .750 export options again.
    • tested update, pushed to my storm-control git branch as storm2_140815 branch
    • could not reproduce jumpy behavior of steve for Hazen this morning, hopefully its done.
    • updated windows on new computer to not allow automatic restart if users are logged in.
    • Dave not issue not fixed (not so bad if first image is conventional. HB attributes this bug to JM. promises yet another version of Dave software coming soon anyway (eee hopefully this is better?).
  • setting up STORM imaging of chromatin-L4E1 (F10, first 100 kb)
    • spots much brighter than the sample 3, which was supposed to have E1-E3 (first 300 kb of F10).
    • Not really as bright as I’d expect though for 100 kb black region. Still not so sure the plate based method gives as good probes.
    • we’ll see how things turn out in the proper analysis.

ChromatinCropper update

  • relabel ‘channel1′ and ‘channel2′ — it’s already hardcoded to grab based on ’647′ and ’750′, so this ‘channel 1′ stuff is just confusing.

Ph Project

  • checking images
  • checking IQR values
  • clarify which panels are which in Fig S1 (wouldn’t have been an issue if we labeled on the figures).

project 2

  • validate and order primers
  • seqs to Jeff to make secondaries for screen
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Thursday 08/14/14

11:00 am – 11:15 pm

Project 2

STORM

  • STORM2 computer swapped to run Hamamatsu camera
  • Hal Steve and Dave all changed dramatically (control software)
  • Previously optimized storm2 branch from other computer NewDaxWriter Fetched won’t run
  • tried merging — still won’t run, can’t record movies
  • tried loading fluidics branch, also won’t run / save files
  • recording old fashion style
  • chrL4 E1 looks decent. Taking O/N STORM data.

PH project

  • some revision correspondence
  • offered equal contribution status.
  • got new cells from Ajaz. Still need secondary antibodies (and blocking).
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Tuesday 08/12/14

11:00 am – 5:00 pm, 8:00 pm – 11:00 pm.

Paperwork

  • started work on BW fellowship app
  • setup account, started filling in general information
  • started new folder in \Paperwork\FellowshipApps\BurroughsWellcome\

Ph Polymerization

  • revising STORM uncertainties section
  • clarified discussion of geometric distributions
  • discussed Xist PRC2 SIM paper

Computer maintenance

  • Cajal teminated remote desktop.
  • messed around with activating licenses, got it to recognize its licenses finally. Did not need to re-enter the keys.

Chromatin Project

  • analyzed E8 E9 data.
  • First 10 movies look pretty decent.
  • There-after the buffer is pretty dead, 750 data quality drops through the floor.
  • I wish these MEA-COT buffers would live longer like the BME-COT buffers. No dice.
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