Sunday 02/16/14

Posted on February 16, 2014 by admin

1:30p – 7:00p, 10:30p – 12:10a

Chromatin Project

Library3 Prep (renewed)

Revised gene list

Code updating

  • Fixed minor bug in BatchLaunchOligoArray: ProcessTimeOut for blastall was in seconds but for java was in minutes.
  • Fixed bugs in GetLocusSenseSeq — had extended to flip genes that are only partially contained within the locus, but hadn’t set the sequence start / end parser to max out correctly at each end.
  • now running as BuildLib3_1401216 (should be 140216).
  • some probe sets badly crash my computer. No idea why. Let’s try using the run in 1kb segment version of the code, I think that was generally more stable, and when crashed doesn’t lose the whole gene, just a bunch of regions. It will be more work to stitch these back together but I’m sure I can write a short script to do it.

STORM

  • Finish O/N STORM of F06-A647 F05-A750. 647 channel at least looks good through the end. see how these 750s came out.
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Friday 02/14/14

Posted on February 14, 2014 by admin

10:00a – 7:15p

Deep sequencing analysis

Effect of Seq Prep on Abundance

  • Prepping samples for sequencing this time at least doesn’t seem to introduce large distortion in abundance.
  • Would have been good to sequence these libraries much deeper though, average read depth on the best sampled is 20 reads. Lots of the variation is in the single count reads. Would be good if everyone were mostly over 100 reads.

EffectOfSeqPrepOnAbundance_lin
EffectOfSeqPrepOnAbundance

Effect of PCR on Abundance

  • these measurements were sadly impacted by the very low concentration of the second duplicate PCR prep
  • (all of which dropped in the two gel extractions after sequencing. Probably mis-trimmed the block. Then weaker bands got harder to see and trim right)

EffectOfPCRonAbundance_lin
EffectOfPCRonAbundance

Reproducability of Abundance variation in probe sets

  • note the ~100 fold variation in relative abundance within a probe set.
  • however almost all the probes are within 10 fold of the most abundant, its just a few that lag out.
  • of the ~250 in the F12 data set, all are present.
  • Seems that the same sequences get over-amplified each time, and the same sequences lag behind.

AbundanceInIndependentProbeSets_F12

Ph Project

STORM

  • 2D imaging, matched COT + BME.
  • imaging WT anti-flag + anti-Ph + anti-Dm0 in 647 STORM with conventional images of BXC and ANTC
  • FISH spots look great. Cluster density looks low, but lets wait till we get a proper dot fitter and can see the contrast.
  • hard to tell colocalization at this resolution.
  • need another beadset before we switch! (need non-3D beads).

New cell staining

  • stain new S2 cells in 561 with anti-Ph to see PcG bodies.
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Thursday 02/13/14

Posted on February 13, 2014 by admin

9:30a – 12:40a

Goals Today

  • Start Bowtie analysis of Deep-Seq data
  • Apply new drift correction approach to STORM data, compare spot quality
  • STORM imaging of PH clusters + DNA FISH
    • if anti-PH fails, try to restain with anti-Flag
    • nuclear signal is pretty weak in PH. staining PH-WT-FLAG cells with anti-flag.

Deep sequencing analysis

  • built indexed lib fasta files
  • Ran Bowtie
  • built matlab bioindexed files
  • converted to matlab fast-indexed structures

More Bead alignment

  • cp2tform (control point to tform) doesn’t do rotation translation without forcing scaling option
  • I made a clone function of this version which accepts ‘rotation translation’ as an input: WarpPoints.m (in TSTORM/Code/Functions on TUCK)
  • Found more issues with FeducialDriftCorrection – cross correlation fails.
    MoreCrossCorrelationMax

crossCorrelationFailure

crosslinked beads

  • 12/05/13 has crosslinked beads — see if these stay put better
  • definetely better stuck. (should quantify);

Drift uncertainties for E1cell2 (unlinked beads)
Distance in nm between matched beads after the warp. sorted in rank order.
implemented using best pair alignment.
47.7950 48.3579 56.3873 60.3818 66.6519
2.4474 4.1889 5.8574 7.7761 8.2402
1.0944 3.7067 28.7937 34.8875 36.6357
0.0742 0.0886 12.5635 15.0466 19.3501
11.5580 11.8013 13.9580 15.5071 17.5983
9.3785 20.3346 22.2668 26.5540 27.4070
4.8066 5.5000 36.0185 40.2484 42.5206
1.0492 5.2735 10.4605 16.4393 52.9124
23.1428 27.3867 68.5654 88.9050 402.9921
35.1398 41.7546 58.7860 64.8746 70.3163
1.4286 5.0899 15.3220 18.8834 24.4787
0.3453 1.2389 12.9271 19.2681 28.1222
8.3024 11.3135 13.4250 31.0762 34.3580
3.6465 13.4411 42.6611 49.2972 52.0924

Typical uncertainties in alignment of 12/13 beads crosslinked to coverslip. (in nm)
0.8811 0.9095 0.9095 0.9095 0.9095
2.5366 3.5464 9.6424 10.3101 11.0085
0.1880 0.3737 1.2259 3.0643 4.8915
0.2896 0.3411 2.6533 2.7363 2.7930
0.1344 0.2061 1.2882 1.6983 1.8770
0.0060 0.0085 0.9791 1.1393 1.7360
0.5859 0.7642 0.8509 1.0569 1.8491
2.4244 3.6952 6.0305 13.0327 13.1788

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Wednesday 02/12/14

Posted on February 12, 2014 by admin

9:50a – 11:59p

Ph Project Cell staining

  • hot wash post-hybe
  • PBS wash
  • Block 1 hour in 2% BSA
  • primary stain rb a-Ph for 2 hrs RT, 6 hrs 4C
  • rinse, wash in PBS, reblock,
  • secondary stains O/N at 4C (m a-lamin 488 + dk a-rb A647)
  • rinse

STORM

  • record max power from all primary laser lines on STORM2
  • imaging dual color F05-A647 F06-A750.
  • initial spots started quite bright, by 11:30p are pretty dim. Probably buffer issues
  • not sure why buffer issues always seem worse / last for less long in dual color imaging. Maybe the low BME?

Exploring feducial drift correction

BeadAveraging2

DriftAnalysis1

It appears averaging the beads and doing no smoothing on the positions gives the finest results.

Integrating on top of 60 frame averaging makes these beads worse (previously thought it was worse without this).
correctedBeads60_integrate5

correctedBeads60

Averaging all bead movies in latest data set (136 movies) at 60 frames combined.

Code changes

  • substantially modified drift correction
  • only integrates frames if the sampling rate = 1
  • if there are more than 2 feducials, it uses the maximumally auto-correlated one as the guide dot, and it’s next most autocorrelated one to estimate drift error
  • if there are only 2 feducials, it uses the bead that moves the least.

This version aligns beads quite tightly.

inMovieDriftCorrected

Aligning beads in sequential images

Align so that two beads match as best as possible (assuming these are the beads that didn’t move at all and compare to these other beads).

beadMatching_displacements

Compare best 2 match vs. best affine match:

beadMatching_byPair

beadMatching_affine

Really does seem like the beads move.

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STORM2 stats 02/12/14

Posted on February 12, 2014 by admin

Max Laser Powers

  • 488 laser head (100%): 580 mW
  • 488 before expander: 275 mW
  • 488 microscope backport: 180 mW
  • 561 at head (2100mW in GUI): HIGH
  • 561 laser at beam expander: 1500 mW
  • 561 laser at backport 900 mW
  • 647 laser at head (1700 mW GUI): HIGH
  • 647 laser at beam expander: 720 mW.
  • 647 laser at backport: 460 mW
  • 750 laser head (2350 mA GUI): HIGH
  • 750 laser at beam expander: 300 mW
  • 750 laser at backport: 115 mW
Posted in Summaries | Comments Off on STORM2 stats 02/12/14

Tuesday 02/11/14

Posted on February 11, 2014 by admin

9:50a – 12:15a

Chromatin Project

Deep sequencing

QPCR

  • run QPCR
  • quantify results from QPCR
  • C:\Users\Alistair\Documents\Research\Projects\Chromatin\Code\Scripts\qPCR_MiSeq2Prep_140212.m

qPCRkappaStandards

MiSeq Prep

  • sample concentrations / calculating dilutions:
  • orig library
    • raw 10 uL scale reaction: 25, 14, 25, nM
    • dilutions 1:10 diluted: 1.98, 2.0, 2.0
    • these are 2x listed concentrations since its a 10 uL not a 20uL reaction volume
  • prep samples for MiSeq run
  • Start MiSeq

Meetings

  • discussion with Jane and Bogdan about chromatin data and models
  • random walks are non-spherical: should look more at elipticity data
  • try log-log plot with 1/3rd 1/2 and 1 lines.
  • schedule STORM users meeting for Monday. Need to organize some lists.
  • Need to measure max laser powers on STORM2 and STORM4 for Monday meeting

Cell staining

Cell prep

  • removed cells from RNase treatment (9p-9a at 37C, hope this wasn’t too much for the delicate structures).
  • SSC treatment. moved to formamide.
  • Prehybed ~1hr at 47C.
  • small hybe oven having issues (shot up to 58 when I removed the three large water chambers).
  • Using large drying oven for better thermal stability

Probes

  1. G01-G02-G03-P1 A647
  2. F03 + F04-P1 A647
  3. G05-P1 + G06-P3 + S1-A647 + S2-A750
  4. G05-P3 + G06-P1 + S1-A647 + S2-A750
  5. F05-P1 + F06-P3 + S1-A647 + S2-A750
  6. F05-P6 + F06-P1 + S1-A647 + S2-A750

Secondary projects

Ph Project

Cell staining

  • (7) S2 cells: G03P2-cy3 + F03P3 + F04P3 + F05P3 + S3-A750
  • (8) Ph-WT 1 cells: G03P2-cy3 + F03P3 + F04P3 + F05P3 + S3-A750
  • (9) Ph-WT 2 cells: D12-cy3
  • all at 0.4 – 0.6 uL of 100 uM secondary + 1-2 uL of primary (each in case of multiples).

OligoPaint project

  • need better explanation of STORM
  • sent fig from XZ review to BB.
  • Also this:

IntuitLocalization

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Goals 02/10/14

Posted on February 11, 2014 by admin

To Do

Ph project

  • analyze gene expression data by chromatin region
    • write code to grab all genes mapping to blue regions
    • take all blue intervals (already recorded), get gene by interval (function already built), let run.
    • the gene matching game! hopefully one of my lists has this covered, otherwise this is going to be a pain as always to match Ajaz’s common names to my common names / gene IDs.
  • Make figures from Confocal data
    • Made preliminary figure from Ph-data (Completed 02/09/14)
    • bodies not highly pronounced. Ajaz gives it the thumbs down
    • Sign-up for more confocal time. Try to image freshly stained S2 cells.
  • make supplemental figure from negative controls
    • no primary
    • Flag in S2

Team project

  • downsample bead data
  • analyze lib4 data

Sequencing

  • prep samples for qPCR (Completed 02/10/14, 11:45pm)
  • Run qPCR (completed 02/11/14)
  • Analyze qPCR data (wrote new script). (Completed 02/11/14)
  • Set up and run MiSeq (Completed 02/11/14)
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Monday 02/10/14

Posted on February 10, 2014 by admin

9:30a – 11:50p

Meetings

  • Group meeting (see notes)
  • Journal club
  • team project meeting

Literature

  • interesting perspective: http://www.sciencemag.org/content/343/6171/596.full
  • Pauli’s paper: discovering new developmental signalling peptide (Science)

Chromatin

Data analysis

  • Analyzed G01-G02 up to image 23
  • start (re-)analyzing F03.
  • F03 data analyzed up through image 16 (34 spots, plenty more to image).
  • F04 data analyzed up to image 22 (33 spots, plenty more to image)
  • F05 data — beads not as bright as bead-parameter settings. Rerunning bead analysis.

Cell prep

  • passage cells
  • plate cells for new staining
  • prepping cells
  • New stains: G01 + G02 + G03 (all 647)
  • F01 + F02 (all 647)

Seq Prep

  • 16 samples, 1:50 dilution, then 1:20 dilution. 4:196, 5:95
  • Column 1: rows 1-8 (A-H): 1:1000 dilutions of samples 1-8
  • Column 2: rows 1-8 (A-H): 1:1000 dilutions of samples 1-8 Replicate
  • Column 3: rows A-H: 1:1000 dilutions of samples 9-16
  • Column 4: rows A-H: 1:1000 dilutions of samples 9-16 Replicate
  • Column 5: 1:10K dilution of samples 1-8
  • Column 6: 1:10K dilution of samples 1-8 Replicate
  • Column 7: 1:10K dilutions of samples 9-16
  • Column 8: 1:10K dilutions of samples 9-16 Replicate
  • Column 9: old library, Replicates in A, B and C, 1:1000 dilution. Replicates D,E,F 1:10K
  • Column 10: DNA standards 1-6
  • Column 11: DNA standards 1-6 replicate
  • 4 ul of sample (diluted sample or straight standard) + 6 uL of qPCR master mix.

Ph Project

Attempting STORM + conv DNA FISH

  • prepping cells for in situs
  • accidently took S2 + 2 WTs (wanted S2 + WT + M). Oh well, let’s see if this works at all for WT anyway
  • running RNase digestion (9:45p)
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Journal club 02/10/14

Posted on February 10, 2014 by admin

Flash Memory: photochemical imprinting of neuronal action potentionals onto a microbial rhodopsin (Cohen Lab). JACS Jan 2014.

High Res confocal microscopy (not STORM/PALM/SMACAM)

  • Two PRL papers
    • High-resolution confocal microscopy by saturated excitation of fluorescence. Fujita et al, PRL, November 2007.
    • Measurement of a saturated emission of optical radiation from gold nanoparticles: Application to an ultrahigh resolution microscope.
      Chu et al, PRL, January 2014.

Intro, review the 2007 work

  • principle: saturation (more laser less response at some point). – this is a non-linearity we can exploit to improve resolution
  • in spatial domain this makes the psf larger
  • in the Fourier domain you get multiple peaks (‘overtone’), more power in these peaks as you increase intensity.
  • find the positions in the image that get overtone are narrow versions of the other.

2014

  • saturate the scattering of gold particles (instead of fluorescence)
  • also look at higher modes, get scattering.

Questions

  • compare to saturated nonlinear SIM?
  • similar prinicple
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Protected: lab meeting 02/10/14

Posted on February 10, 2014 by admin

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