Monday 01/20/14

Posted on January 20, 2014 by admin

9:40a – 1:00a

Goals

  • send fig files to Jeff
  • Upgrade memory on Cajal.
  • Image ANT-C complex
  • analyze black K100 (internal black domain)
  • plots of internal vs. external.

Lab Meeting

  • see notes.
  • journal club notes

Computer stuff

  • Memory installed on Cajal. Need to upgrade Server edition to use more than 32GB of RAM.
  • Windows 2012 Server Standard will do it, and can be upgraded directly from 2008 Server R2 Standard.
  • Link to licenses from Newegg for multiple remote desktop for 2012.

matlab-storm development

  • weird issue with imagesc not printing indexed data to figure windows seems to have gone away.
  • added colormap selection options and tested contrast adjustment in ChromatinCropper

adding colormap selection to STORMrender

  • To Do: add drop-down menu next to levels to select color for the channel

Chromatin Project

Presentation

  • working on slides for meeting with XZ

STORM

  • imaging ANT-C on STORM2
  • fresh glox, 2uL MEA 5uL COT, excellent switching (also a brightly stained 400 kb locus).
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journal club 01/20/14

Posted on January 20, 2014 by admin

Sleep clears metabolites from the adult brain

Colenso lab journal club

  • Consensus that sleep is restorative, little mechanistic understanding of why.
  • Lymphatic systems does not extend into CNS / brain. (in other part of the body used to clear fluid)
  • spinal fluid flows around the space around the arteries in the brain.
  • Observer much more diffusive flow into the brain during sleep than during awake.
  • Interstitial space increases during sleep or anesthesia, allowing more flow.
  • Mediated by osmotically triggered change in cell size. – transfers water from the artery into the interstial space.
  • block arrousal by blocking adrenergic signaling in awake animals.
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Protected: lab meeting 01/20/14

Posted on January 20, 2014 by admin

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Sunday 01/19/14

Posted on January 19, 2014 by admin

10:00a – 11:40a, 1:30p-6:30p,

  • Prepare submission for Drosophila image competition

chromatin project

  • adding DAM-ID data and ChIP-seq, and FARIE-Seq to chormatin colors overview
  • working on slides for meeting with XZ
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Saturday 01/18/2014

Posted on January 18, 2014 by admin

10:30a – 7:20p,

matlab-storm

STORMrender online

  • Getting Jeff set up with STORMrender

debugging:

  • STORMfinder added paths in initialization, allowing matlab-storm to access depreciated versions of code in folders that weren’t supposed to be on filepath

Features to add to STORMrender

  • user defined colors
    • make a little RGB GUI, enter 3 numbers, show a swath patch of the color
    • automatically populated with each of the loaded channels (STORM, overlays, Z-colors per STORM).
  • automatic auto-contrast for overlays

Bugs to address

  • log-scale tick box doesn’t work
  • Levels doesn’t automatically populate until you select the slider.
    • Should trigger the update triggered by dropdown menu selection when the image is loaded.
    • This should happen for overlays as well as STORM images (though I guess you never just load an overlay).

Learning Matlab Debugger

  • when stopped in debugger, matlab workspace changes to be inside the function.
  • step just progresses to the next evaluation statement.
  • step-in opens internally called functions, opens these functions in editor and lets you see their workspace
  • add new breakpoints and hit continue to run through loops etc (don’t want to step through these one at a time).

Chromatin

New metrics

TADS

  • numTADs is not so useful (just counting boundary domains, BDs). Some domains are essentially all a single TAD and happen to pick up one or a pair of TAD boundaries at each end of the domain.
  • really want a weighted sum. e.g. 5 TADs of equal length should score a 5. 5 TADs dominated by 1 big domain and 4 tiny ones should score close to 1.

STORM

  • reimaging of F04-647 F03-750
  • confirmed switching is much better in MEA, should repeat imaging this locus
  • too late at night now to set up run (1:00a).

4C broken again. Up to 11.6 C. Waiting for maintenance to arrive.

Ph Project

  • staining Pc cells and new antibody-screen cells with Dm01-488 to help address the nuclear / cytoplasmic issues.
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Friday 01/17/14

Posted on January 17, 2014 by admin

11:50p – 12:45a,
2:00a – 3:20a (remotely)

Chromatin Project

Data analysis

Goal: Sort data by expression levels.

Getting FPKM expression values:
Downloaded data from NCBI GEO database from the 25 Drosophila Cell lines paper.

Downloading gene name conversion tables from FlyBase
ftp://ftp.flybase.net/releases/FB2014_01/precomputed_files/genes/
This file is good: fbgn_annotation_ID_fb_2014_01.tsv. My earlier gene list has issues. No Antp.
CGs aren’t fixed length. Antp is CG1028, but lots of other genes are CG1028#. Uses strcmp.

Lots of new plotting stuff for libraries

Here’s a few examples, working out plotting genes and visualizing gene expression levels.

MapsWithGenesAndExpression
mapWithGenes
GeneProperties_label
GeneProperties1

Some issues that need to be fixed still:

  • match area names to table. This should be easy, original table is alpha-numeric. Only problem is the conjugate domains and the stuff from the previous MycroArray and LC libraries…
  • GetLocusColor is buggy. Really just need to take the difference between the locus Start and locus End and the nearest boundary and record the chromatin type there. Not good enough to bounce out to the nearest interval boundary, this is less accurate and can severely distort things in cases.

STORM

  • made fresh 1M MEA
  • 2 uL MEA + 5 uL COT much much brighter A750 switching than previously. Need to repeat previous images with this stuff. Might need to always make MEA fresh (takes maybe 150 s).
  • Bead concentration getting a bit low, time for new 1:1000 beads.
  • stage movement is still cr*p. Need to manually pick regions until I can find time to get the USB interface to the nano Z-positioner working.
  • sample imaging around 8p
  • by the time the sample is arrayed and all the conventional images are taken, the buffer seems to have given out?
  • 10:30p, buffer basically depleted — 750 switching is completely crp now.
  • Maybe not — 2nd position looks better, maybe forgot to delete the calibration position from the position lists… beads were pretty dead there too…
  • okay, switching alright still (12:35a). I’m going let this run overnight and hopefully the data’s okay. At least we have on-the-fly compression of the bead data now!
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Thursday 01/16/2014

Posted on January 16, 2014 by admin

9:30a – 1:00a

Happy Birthday.

Goals

  • Stain new cells: ANT-C, G05, D12
  • chromatin probe synthesis debugging
    • order new Maxima and T7
    • create list of probes to make next
  • start compressing bead data! – postponed again.

Chromatin Project

  • Finish O/N STORM of F04-A647, F03-A647
  • start analysis of F03,F04 and F04,F03 data
  • Finish running ChromatinCropper on F10 data
  • update and reboot Monet
  • Working on slides for next meeting with XZ: full Lib2 data update

Coding Genome analysis

  • adding gene viewer: GetLocusGenes.m
  • now built into the LoadKc167Data.m
  • Note current version of flip gene only flipped complete genes, not genes that edged into the region.
  • Fixed in GetLocusSenseSeq.m
  • constructed maps for all of Lib2

Coding matlab-storm

  • Multi-color display works fine.
  • single color loading of demo data fails.
  • fixed single color loading — imagesc won’t write indexed images (i.e. 2D matrices, not RGB data) to axes in a GUI (I just get a solid colored field. I can change the color, but not actually get the image). Sending the same command to the GUI handles.axes2; images(I); and to figure(1); imagesc(I) back to back in the same code produces the image correctly in the figure and as a solid field in the axes. As soon as the image is an RGB image it displays correctly in both. WEIRD matlab. weird.
  • (simple work-around, use ind2rgb(I,map) to convert first. Only it’s pick about the range of the map, the data type of the map (needs a double, even though you can call a color map on a uint16, it gives weird results). Also ind2rgb takes uint16 but returns double, which doesn’t seem obviously scaled, so I use makeuint to convert these to balanced contrast uint16.
  • at some point I want to get rid of the automatic balanced contrast, or make it a toggleable function rather than an intrinsic property.

Ordering

  • ordered new Maxima (full length)
  • ordered new T7 kit
  • ordered new ProRAID (4 bay) — easier to fit on desk, no adventage with 8 bay sincy they max out the RAID 5 array at 4.
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Protected: Library 2 results summary so far

Posted on January 16, 2014 by admin

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Protected: chromatin snapshots 01/15/14

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Wednesday 01/15/14

Posted on January 15, 2014 by admin

9:30a – 12:30a

Chromatin analysis

  • loading Bogdan’s black area data.
  • units or normalization is off somehow. These clearly don’t line up as expected.
  • Redoing the spot-processing myself. Working on F1 data (very high background, this is a bit frustrating).
  • F10 data looks pretty good, low background
  • see data
  • continuing work on Library2 summary, see post

Tuck RAM update

I can’t turn the computer off at the moment because people are using it. I had previously booked a few hours this morning to turn the machine off and try to install the RAM. here’s what I tried during that interval:

I first installed the six new 8 GB modules in the adjacent positions of the existing six 4 GB modules. Upon booting, if I check computer properties, the system says 72 GB of RAM, 23 usable.

I then removed the 6 old 4 GB modules and installed the new 8 GB modules in their place (these memory card slots have a blue plastic trim instead of a black plastic trim like the other 12). The system still boots fine. Now it says 40 GB of RAM, 23 usable.

I will contact all the users and try to arrange an alternative time when I can freely turn the machine off to shuffle the positions of the modules if necessary and check the BIOS. If you let me know what times work for you, I’ll try to find times that work for all the other users to cease their computation and for me to be free to work on troubleshooting the RAM install.

Alright, looks like this is going to get delayed a month or more, rebooting Tuck is not acceptable. I suppose we can still stick the old DIMMs from Tuck on Cajal.

storm-control update

  • debugged my new imagewriters.py for STORM2.
  • python / numpy uses matlab style indexing for images (Height by Width), so you need (ypix,xpix) not (xpix,ypix) in reshaping arrays.
  • fixed bug resulting from previous failure to rezero the average image. Now initialized as zeros.
  • still need to commit my changes on STORM2 and push back to my personal branch.
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