Protected: project 2 analysis 07/02/14

Posted on July 2, 2014 by admin

This content is password protected. To view it please enter your password below:

Posted in Genomics | Tagged | Comments Off on Protected: project 2 analysis 07/02/14

Tuesday 07/01/14

Posted on July 1, 2014 by admin

7:30 am – 12:25 am

Goals

  • finish probe making
  • compute scaling of sub-paths through condensed sticky globules
  • revise fig 2 for oligo secondaries
  • write caption for fig 2 for olgio secondaries
  • write new discussion section corresponding to fig 2 for oligo secondaries.

Oligo secondaries paper

  • working on figure 2
  • wrote draft caption for fig 2
  • working on revising main text.
  • working on supp fig 14
  • working on XZ’s revisions round 2

Probe making

  • label probes,
  • test stain new cells with D08-D12-p3

Meetings

  • send email to LM
  • meeting with Anton, discuss preventing polymer explosions — see notes
  • send Matlab Steve viewing code to Hao

Cell staining

  • new stains,
    1. C02-P1-405 + S1-A647,
    2. E05-P1-405 + S1-A647,
    3. D08toD12-P3-405 + S3-A647 1.5 uL each probe
    4. L2F03-05 P3-405 + S3-A647 + L2F06-P1-405 (40 uL) 1.5 uL each probe

Coding notes

  • running MirnyStickyTemplateV6 with (global) energyMinimization, variableLangevin integrator, and SmoothSquareWellTailedForce for specific attractions.

    a = SimulationWithBonds(timestep=20, thermostat=0.05)
    a.setup(platform=platform, verbose=True, GPU=GPU,integrator="variableLangevin",errorTol=.0001)

    SimulationWithBonds inherits from Simulation and I’ve just added my own potentials here rather than modify openmmlib.py.
    Does it matter what I put for timestep if I’m using “variableLangevin” integrator? Have I specified the error tolerance for the variable integrator correctly?
Posted in Summaries | Comments Off on Tuesday 07/01/14

openMM simulation notes

Posted on July 1, 2014 by admin

Recommendations from Anton for avoiding exploding polymers

Initial recommendations

  • integrator choice:
    • variableLangevin integrator recommended
    • takes an error tolerance instead of a timestep variable (e.g. 10^-4)
  • thermostat = high values high friction
  • mass_scale = argument of init of simulation (see openmmlib.py)
  • attractionRadius (be careful about interaction over other polymers).
  • wiggle_dist = higher more oscillation, less chain crossing.
  • localEnergyMinimization(maxIterations=1000,tolerance=1)
    • local energy minimization with attractive potentials might lead to super-position.
    • Try global energy minimization.

conlen = 1 nm * length_scale (=1).

potentials

  • smooth square well force
  • polynomial force
  • sticky particles should be extra hard.
  • every particle has a charge (sticky yes no) repulsion strength yes no.
  • extra repulsive force
  • try hard-core forces (very large potential energy at overlap).

dynamic adjustments (changing parameters during simulation)

  • things of interest to change: friction
  • crude way, use output as input
  • system block
  • try energyMinimization in place of localEnergyMinimization
  • avoid any collapsed pairs (they just get worse). — this is time to quit
Posted in Chromatin, Software Development | Tagged | Comments Off on openMM simulation notes

Monday 06/30/14

Posted on June 30, 2014 by admin

10:00 am – 5:45 pm, 10:00 pm – 1:15 am

Meetings

  • group meeting (see notes)
  • journal club (see notes)
  • discuss probes with George
  • setting up meeting with AG to discuss code
  • discuss with XZ Brian’s paper and revisions
  • project 2 team meeting discuss data analysis

Coding

  • integrate new cluster analysis code into project 2 core analysis scripts

Probe making

  • set up RT reactions
  • run diagnostic gel. — clearly have RNase problems substantial smears and reduced incorporation.
Posted in Summaries | Comments Off on Monday 06/30/14

Journal Club: B-Actin mRNA in neurons (Singer)

Posted on June 30, 2014 by admin

Science

Basic observation

  • chemical stimulation of neuron, see more B-actin mRNA in axon (2x)
  • also see more B-actin mRNA following gentle protease treatment — propose mRNA was masked
  • do transcription controls and trafficking controls
  • block neural stimulation pathway, also don’t see more B-actin
  • kinetics of response (detected in less than 10 minutes). within 20-40 min has returned to masked state. (half-life tau = 7 min)
  • is this long enough to make bigger spines? Well certainly if you have multiple stimulations
  • is more actin actually made? Use dendra-2-B-actin photo-conversion to detect newly synthesized B=actin.
  • rRNA detection also increases following protease treatment
  • masked B-actin (revealed by protease) associate more the rRNA granuales (revealed by protease).
  • bright ribosomal structures have reduced mobility, become more mobile following cLTP
  • live imaging of B-actin mRNAs observe faster diffusion in stimulated cells.
Posted in Journal Club | Comments Off on Journal Club: B-Actin mRNA in neurons (Singer)

Protected: lab meeting 06/30/14

Posted on June 30, 2014 by admin

This content is password protected. To view it please enter your password below:

Posted in Lab Meeting | Comments Off on Protected: lab meeting 06/30/14

Sunday 06/29/14

Posted on June 29, 2014 by admin

10:30 am – 3:00 pm, 6:00 pm – 7:00 pm, 8:30 pm – 11:30 pm

STORM

  • finish imaging C11

Cell prep

  • passage newly plated cells to 75cm2 flask
  • fix cells, prep cells for staining

Probe making

  • run RT PCR for D08 – D12 + neg cntrl
  • run PCR cleanup
  • run IVT reaction

Chromatin Analysis

  • Analyzed B10 up through image 23
  • Analyzed B11 except last 3 images (7, 8, 9)
  • Analyzed B12 through image 4
  • Analyzed F11toF01 through image 26 (50+ spots)
  • Analyzing F01 nearly same size as F11 to F01 ( through image 31, 43 spts)
  • Analyzing E12 much smaller than F01 (despite 2x more DNA, 10kb vs 5 kb),
Posted in Summaries | Comments Off on Sunday 06/29/14

Saturday 06/28/14

Posted on June 28, 2014 by admin

10:45 am – 5:30 pm, 9:00 pm – 11:50 pm

Small tasks

  • restart data copying after computer freeze last night (GPU computation abort failed)
  • restart sticky polymer simulations

STORM imaging

  • imaging sample C10 (blue 33 kb vg locus).
  • Conv images nice and bright
  • images look pretty good.
  • STORM imaging sample C11 (black 10 kb)

Meeting with Ajaz

  • follow up to ANTP PH cluster analysis: BX-C and control region
  • Ajaz found new potential polymer modeling collaborator (see email)
  • will get manuscript to XZ

Chromatin Data analysis

  • Analyzed D01 data (ChromatinCropper)
  • Analyzed D02 data
  • what happened to D03 data?!
  • Analyzed D04 data
  • Analyzed D05 data
  • Analyzed D06 data (way too large, not sure what’s up with this locus!)
  • Analyzed D07 data
  • black trends looking decent

Cell culture

  • passaged cells yesterday
  • start new cultures from frozen stock today
  • density looking pretty good, should be able to plate a batch of these cells tomorrow and passage the rest.

Chromatin experiment planning

  • ordered toe-hold displacement probes to try to remove un-bleached, un-imaged secondaries for sequential hybe approach.
Posted in Summaries | Tagged , | Comments Off on Saturday 06/28/14

Friday 06/27/14

Posted on June 27, 2014 by admin

10:45 am – 11:00 pm

Prep for meeting with XZ: model summaries

  • rosettes actually have very complicated scaling
  • generally sticky nodes seems to scale better.

ScalingRosettes2

ScalingRosettes1

ScalingStickyPolymer2

scalingStickyPolymer

internalScalingStickyPol

Conclusions from meeting with XZ

  • First priority: get at least a first look at the internal scaling of black domains
  • Second priority: talk to Hazen and Steven about demoing commercial adaptive optics (improve 750 imaging deep in cells?)
  • start discussion with Mirny group

Oligo secondaries

  • working on figure
  • find some regions to do cross-sections?

STORM

  • finish staining C09, C10, C11 (hot washes)
  • imaging C09
Posted in Summaries | Comments Off on Friday 06/27/14

Thursday 06/26/14

Posted on June 26, 2014 by admin

9:45 am – 1:30 am

Chromatin Modeling continued

Checking my simulation results from the polymer models I was running last night.

Simulation Results

BBv1 no attractive potential does give expected scaling:

BBv1_freePolymer_works

Adding an attractive potential does increase clustering. Has more effect on large polymers.

Notes from meeting with Max

More coding

  • Fixed up openmm-polymer code
  • implemented extension script to import and build off the openmmlib (I called my function lib openmmlibExt).
  • with some difficulty (stupidity), finally implemented node specific interactions
  • simulating free polymers,
  • simulating uniformly sticky polymers
  • simulating periodically stick polymers

Primer ordering

  • Lib4 primers still had the same bug as the Lib3 primers. Now when back and editted old scripts to fix this bug (will be logged in GitScripts).
  • bug was in the export list of T7 rev primers, the sequences of the Fwd primers was used and not the rev primers.
  • wrote new functions to index 96 well plates and to convert fasta files to csv tables for ordering primers.
  • validated primers in quick test.
  • remembered (belatedly) to add T7 to project 2 L5 primers and strip off extra Gs
  • sent primer orders to Alec.

Staining

  • new cell stains: C09 C10 and C11 (tiny blue and black regions)
Posted in Summaries | Tagged , , | Comments Off on Thursday 06/26/14