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Oliver Rando: I: Structural biology of the yeast genome II: Mechanical disassembly and reassembly of mammalian reproduction

MNase digestion — nucleosome phasing
26 different histone modifications mapped genome wide. in 20kb of yeast chrIII

chromosome conformation capture
mapping 300 nm

the 30 nm fiber mode

  • solenoid model, regular 30 nm model
  • zigzag, 3 nucleosomes / turn, less regular
  • non-existent in vivo, only in dilute in vitro preps
  • using MNase to map nucleosome-nucleosome contact maps.
    • different fiber folding predict different peaks
    • challenge, need to repair ends of MNase digested DNA while crosslinked to allow ligation
    • claim: Nuclesome resolution HiC yeast map. semi-blocky structure on scale of 100 nucleosomes.
    • clear relation between local gene structure
  • in yeast squares on scale 2kb
    • is this really contact or is it co-accessabililty?
  • difference between triangle and mountain
    • triangle structure are highly expressed.
    • tandem orientation genes more likely in same block.
    • divergent genes, clean break at promoter.
    • (I suspect this is all transcription driven not topology driven)
  • no evidence for regular 30 nm fiber
  • ‘Gene loops?’
    • +1 touches N-2 nucleosome etc maybe just as well or better than the N.
    • ‘gene crumples’ rather than ‘gene loops’
  • access to data about DNA as a chromatin polymer

Pt II: inheritance of acquired characteristics

  • compare high protein vs. low protein fed males
    • hundreds of (liver genes) can distinguish the difference between dads
    • upregulated genes enriched in cholesterol and lipid biosynthesis
    • downregulated are nothing in particular
    • see phenotype differences as well.
  • other work
    • starve males in utero, up-regulate glucose and colesterol
    • in utero during dutch hunger winter — increased diabetes etc. bodies hoarde calories
  • connection between metabolic phenotype and later generations
  • hypothesis ‘sperm epigenome?’
  • also data showing molecules in the sperm fluids
  • artificial insemination doesn’t work in mice. Need to do IVF.
  • phenotype less penetrant than natural mating but still passable. Can sequence the rest of the sperm sample or the blastocyst. Look for cytosine methylation patterns.
    • these methylation patterns and histone modifiation patterns don’t seem to carry the info.
    • small RNAs?
  • small RNAs (under 40bp).
    • types: 70% are tRNA fragments. Also 19bp RNAs and microRNAs.
    • most abundant form is 25-30% of all small RNA in sperm
    • see differences in these guys in males with different parental effects
    • tRNAs directly linked to metabolism, so this is a reasonable messanger. The particular ones here are logical choices.
    • tRNAs are used in retro-element replication. (e.g. HIV packaging)
    • tRNAs prime reverse transcriptase for example. Also true for endogenous retro-elements
    • can out-compete other
  • are these refuse from earlier sperm metabolism or functional?
    • charged testes tRNAs don’t correlate with deitary effects of sperm on tRFs.
    • testes small RNAs vs sperm — testes don’t make lots of tRNA fragments. more abundant in sperm (50 fold).
    • tRNA fragments accumulate during sperm maturation in epididymis.
    • deep sequence somatic cells from epididymis, find lots of these. (transfered from soma to germline?)
  • Sperm maturation
    • change lipid composition
    • gain 200 proteins (despite being translationally inactive) shipped in vesicles from epididymis.
  • isolate and sequence epididymesomes, similar tRNA profile as mature sperm.
    • mostly come from distal epididymis, not proximal.
  • do immature caput sperm have tRNA fragments?
    • yes but less than mature ones.
  • Reflections
    • increasingly find other examples of soma-germline communication
    • mostly mediated by small RNAs
    • evidence against this hypothesis? Find high levels of intact tRNA in caput sperm.
  • follow up
    • genetically express modified label tRNAs in somatic cells, look for these in germline.
    • make library of tRNAs express in virus, look for trasnfection into germline cells.

Biogenesis

  • Epididymisis as a sensory organ for gamete RNA engineering
  • try to squirt in epididymesomes into oocytes.

consequences

  • knockdown GlyC tRNA, see strong overexpresseion of ~30 genes.
  • genes that are de-repressed in knockdown are repressed in low protein sperm.
  • All 25 genes are regulated by MERVL, an endogenous retro-element
  • MERVL uses tRNA leucine (not Gly) for its replication. odd.
    • turned on in 2 cell totipotent stage. Turns off in 4 cell stage and back on in 8 cell stage.
    • in an ES cell colony, 1:100 cells is oct4 negative and is MERVL positive, cells cycle in and out of this state.
    • FACS these cells and implent. MERVL negative cells are pluripotent (can’t make extra-embryonic tissues)
    • MERVL cells are totipotent, also make placenta.
  • phenotypes seen are similar to uterine implantation phenotypes. Possible model:
    • tRNAs affect implantation and placenta vs embryo growth?
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Monday 04/14/14

9:45a – 9:00p

Computer issues

  • Attempted to swap memory into Cajal.
  • Cajal won’t boot up with any of the memory
  • restored Tuck to 6x 8GB DIMMS.
  • Cajal has 12x 4GB DIMMS
  • wrote to IT to help get system back up.

PH project: requests from Ajaz

  • pick some poly-lysine tonight around 6:45pm?
  • Below are the things which need to be adjusted for main model figure. We need to reorganize the model figure (no. 6). In this figure we need
    1. two schematics for models
    2. plot showing fraction clustered as a function of PH-ML conc.
    3. Representative images
    4. Plot showing diameter varying as a function of PH-ML conc. OR distributions of cluster diameter at different conc. of PH-ML as you plotted for low, mid and high PH-ML concs. (Here I wonder if we show plot for cluster size vs PH-ML conc. we might need to run simulations to show how diameter changes as we increase the PH-ML conc. Something similar to what we have for fraction clustered vs PH-ML conc. plot)
    5. Please see Bob’s comments regarding this figure also. (in the manuscript revised by Bob) According to him schematics are not easy to follow and simulated lines should be smoother (which I think means running the simulations many more times)
  • Actually let’s put this on hold. Currently redrafting paper into shorter 4-fig version and I don’t think there will be time or space for the modeling.
  • still should run PSC clustering data.

Probe Making

  • run oligo-clean and concentrate of probes
  • quantify probe concentrations

E5-E12F1-F8_probes

Troubleshooting labeling

  • Re-measured temperature of hot-plate.
    • setpoint 88C, read with block inverted, thermometer on top, now 78C. Used this for hybes.
    • setpoint 85C, read with block inverted, thermometer on top, temp 71C.
    • setpoint 88C, read with block right-way-up, embedded thermometer, 81C
    • setpoint 85C, read with block right-way-up, embedded thermometer, 78C

New stains

  • C07 treat with 2x SSCT 50% form along with other cells when they come out of RNase. Restain with E04 probe. Denature at usual 85C (glass temp ~78C).
  • new cells RNase treated, extended prehybe at 47C. stain with C01. Denature at usual 85C
  • new cells RNase treated, extended prehybe at 47C. stain with C01. Denature at 100C (glass temp will be 95C at best)
  • Aim / hope: RNase treatment decreases background from expressed locus, extended 47C prehybe increases suspetability to melting during denaturing step.
  • hope 2, change in hot-plate as source of problem now fixed?
  • old cells, RNase treated, stain with new E06 probe (1.5 uL of 2,000+ng/uL probe + .5 uL primary)
  • old cells, RNase treated, stain with new E07 probe (3 uL of ~800+ng/uL probe + .5 uL primary)

OligoSecondaries

  • working on methods section for paper
  • sent current working draft back to Brian.

Review

  • finished checking equations, found small error in my calculations. Still don’t match authors calculations.
  • finished writing up review comments and submitted review.
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Saturday 04/12/14

9:30a – 4:00p

Computer Management

  • attempting to give users rights to share their hard-drives on the network without having full Admin capabilities to do anything they want.
  • Tried adding my ‘User’ account to these groups
    • “Performance Monitor Users” — can access performance counter data locally and remotely.
      • This doesn’t actually let me launch task manager without Admin rights. Not too surprising, since I can kill tasks from task manager.
      • Can’t even access the windows program “Performance Monitor” without Admin rights.
      • Can’t launch the “Resource Monitor” without admin rights either
    • “Access Control Assistance Operators” — windows / google seem to provide no information on what this really does.
  • Can now open preferences, still get asked for admin password to try to change advance sharing file permissions

Chromatin Project

Data Processing

  • SplitDax finished (hopefully for the last time) on E02 data
  • running bead averaging / compression on E02 data
  • DaoSTORM finished on E03 data

troubleshooting hybes

  • have staining now of C01 in both new cells and old cells
    • looks better in new cells
    • neither is comporable to earlier stains or restained c8 cells.
    • some cells have strong/decent stains and some cells lack staining.
    • pretty sure there’s higher background, different quality. May relate to lack of RNase treatment in new cell round. —

NewCellDecentSTORM NewCellDecentSpot PrevGoodC08cellC01restain C08C01_matchedLaser oldCellNewBuffer

More notes

  • should have checked — C01 is 240 kb Y. We probably do have mRNAs in cytoplasm.
  • don’t have spots in every cell, when we do have spots the cytoplasm is dimmer. maybe I’m just staining RNA. Can treat these cells and see if we lose stain?
  • previous C01-C08 batch of cells were first imaged 1 day after I made new RNase solution, so I don’t have it for sure in my notes but I’m pretty confident these guys didn’t skip the RNase step. So current stains are consistent with not denaturing the DNA, except that a side by side re-treatment of C08 cells does denature for strong incorporation. So something is still different in the cell prep that affects the denaturation, rather than the denaturation itself.
  • maybe RNA also dilutes out concentration of local probe (I kinda doubt it, probe concentration is pretty high since whole hybe soln is visibly blue).
  • maybe somehow cells are over-fixed and won’t denature? I think I’m very consistent though on fixative and fixation times…
  • I guess I often let them sit longer in the glycerol than the 30 min I have been doing — I sort treated that as a minimum to get properly equillibrated rather than an exact time step.

To try

  • RNase treat current stained C01 new cells, see if foci are lost. Treatment started 1:20 pm.
  • RNase treating new cells prepped with new buffers. Will then do extended pre-hybe.
  • 95C denature of RNase treated cells prepped yesterday and restain with C01.
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Friday 04/11/14

9:30a – 10:00p

Goals

  • Need to finish review
  • Need to make slides for symposium at Penn
  • send revised
  • Troubleshooting sudden failure in staining

Troubleshooting staining

Background of problem:

Stained new cells on Saturday, went to image these on Monday — no staining. Suspected extended hybe time could have been problematic, or something wrong with new multi-color probes. One idea I had about the extended hybe time was that the rubber cement seal is not very good and there’s a fair amount of condensation onto the slide, which may have leached out the probe concentration. I tried staining another three coverslips of cells using a probes that had previously worked and using a dry box. I still observed condensation and I still observed no staining. Now I thought I might have screwed up the cell prep, so I repeated the cell prep with a fresh batch of cells on Wed and stained these with E3 probes for which I very recently got beautiful staining. Yet again, no staining. To really see if this was hybe buffer / staining conditions or cell-prep, I took an old coverslip (previously stained for C8, a very small weak staining region) and stained again with the large / strong C1 probe. I used this same probe mix and hybe buffer to stain some of the cells prepped Wed, one with and one without RNase treatment. The C1 on the old cells stains great, the new prepped cells both no staining.

The cell prep is reasonably straightforward and I had been using all the same buffers and stuff for a while, so I’ve no explanation of why this should suddenly fail. I did notice a little growth in my PBS glycerol, which I promptly replaced. Yesterdays cells were briefly exposed to this before I noticed the tiny cloudy clusters in the glycerol. I have a hard time believing though that this brief exposure to some extra bacteria or mold eating the glycerol would completely and utterly destroy the ability to hybridize FISH probes to the genome…

All New Buffers

  • made completely new PBS
  • made new 1/2x PBST and 5% formaldehyde
  • made new 0.5% Triton-X in PBS
  • have new 20% glycerol in PBS (using old stock of 10x PBS though…)
  • rinsed PBS squirt bottle. Maybe we should not use it for this round just to be sure.
  • made new 0.1 M HCl from 37% stock

Area cleaning

  • bleached and washed bench
  • bleached and washed pipettes
  • new pipette tips
  • set up new clean-zone at desk with bench guard.

New Cell Culture

  • started new cell culture from frozen stock.
  • Probably enough cells here to plate 1 well and try staining? They may be a little pissed just coming out of LN2 today, but it’s a comparison.

New cell staining controls

  • Resuspend new cells and plate in 4 wells
  • Resuspend old cells and plate in 8 wells
  • allow cells 1 hour to attach
  • follow my protocol
  • stain new cells with C1 probe (no RNase)
  • stain old cells with C1 probe (no RNase)
  • stain new cells (with RNase 32 min at 37 treatment) with E4 probe. This sample kept in separate dish for this step.

More control ideas:

  • RNase treat C7 and then stain it with C1. (see if RNase treatment is a problem / contaminate). It is a new batch of RNase dilution (same original enzyme lot).
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Additional Windows Server 2012 notes

On Windows Server 2012, domain users are not allowed to log on locally by default.

To edit this, follow these directions. Add Domain Users.

Can also edit other permission logs per user here.

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Thursday 04/10/14

10:15a – 11:00p

Presentation Prep

  • XZ wants to meet at 3P, need to make slides (for project 2)
  • see more project 2 protected notes.

Troubleshooting stains

  • sample E03 (previously stained beautifully) failed to work on new cells.
  • Not a simple omission of something in sample prep.
  • Old C01 stains look great. Not a buffer issue or other microscope issue.

New stains

  • old cells (previously stained with C08, dim stain, should also washout during hybe). Stain with C1, 1.5 uL.
  • new cells + RNase, Stain with C1 1.5 uL
  • new cells – RNase, Stain with C1 1.5 uL
  • dilute everything in old “hybe mix” (same buffer, different aliquot than used yesterday).
  • prehybe in new 2X SCCT + 50% formamide made yesterday.
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Wednesday 04/09/14

9:30a – 8:30p

Chromatin labeling

  • stains failed again. No stains yet detected with this batch of cells.
    • maybe it’s a cell prep issue? This F07 probe worked fine before.
    • maybe some reagents went bad in the cell prep? Make new formamide and hybe buffer

Probe Making

qPCR followup

  • Run on new 2% gel 2.5 uL of the second elution of DNA from the limited-cycle PCR
  • Melted gel. Should remember to always change 100% of the buffer and let’s go back to 10 instead of 11 min.
  • lanes: Lib3 E05-E12 (top), F01-F08 (bottom). Also showing yesterdays gel on old gel (not a good week for gels!)

E5-E12_F1-F8 melted_qPCRgel

RT reactions

  • individual reactions: 10 uL RNA + 4 uL primer + 4 uL RT buffer + 2 uL dNTPs + 1 uL inhibitor + .1 uL
  • all prepared with P1 primer
  • Master mix: 18*4 uL primer = 72 uL
  • 72 uL primer + 72 uL RT buffer + 36 uL dNTPs + 6 uL Maxima enzyme + 18 uL inhibitor. 10 uL each, add to 10 uL RNA.
  • out of dNTPs, thawing new dNTPs
  • running RT reactions
  • using last 2 pre-cast gels. Ordered more gels today.
    • also ordered more T7 high yield quick mix kit
    • more DCC-5 columns

lib3_E05-12F01-F08probes

Troubleshooting Cajal Server 2012 Insight compatability issues

  • InsightM gives “Frame out of range!” pop-up error when passed region parameters on Cajal windows 2012.
  • This error requires the user to press okay. There-after it seems to analyze just fine. This includes processing the ROI correctly. Batch analysis is suspended until the OK is pressed — very disruptive to automated processing.
  • copied files to Monet, run fine using the exact same InsightM version.
  • wrote to Bo to comment on bug.
  • Meanwhile commented out the ROI parameters on system run in Cajal local.

Cell staining

  • passaged cells
  • created new 75 cm^2 flask of cells at mid-density for Bogdan.
  • plated cells — reprep new cells for hybes. See if a possible cell prep issue is why my hybes suddenly started failing?
  • New Hybe Dilution Mix, 10 mL, 1.4X
    • 2.8 mL 50% dextran sulfate
    • 1.4 mL 2X SSC
    • 6.5 mL DI formamide
    • 100 uL 10% Tween 20
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Tuesday 04/08/14

9:30a – 11:00p

Goals

  • reschedule Dentist
  • try 2, Cajal windows upgrade
  • Need to finish review / referee paper
  • Set up STORM imaging of E3
  • Test new hybes
  • Finish writeup #2 draft for XZ. sent back to team for comments
  • Start writeup #3 for XZ.
  • Meet with Steven to integrate writeup #3

Probe Making

  • qPCR of E5-E12 + F1-F8
  • picked off samples at 2500 – 3000 RPU
  • ran on old gel. Should re-run on fresh gel.
  • Attempting column cleanup using 96-well plates and multi-channel pippettors
    • this is definetely less hands on.
    • and I think per column its cheaper. And spatially smaller
    • spinning at 3000 x g, with open lid columns.
    • TROUBLING: eluted with 12 uL ddH2O, recovered less than 5 uL.
  • setting up in vitro reactions with quick mix.

Cell Staining

  • observed that even without water in the bottom of the box the rubber cement still appears lose and hydrated after O/N stain.
    • Maybe next try keeping lid open / putting slides into oven straight, no box.
    • Maybe skip rubber cement completely? or try a mock without cells, just a coverslip and hybe soln (no probe)?
  • completed hot washes for stains made last night.

STORM

  • imaging E03 sample
  • ran out of time, did not set up new multi-color sample. Will test tomorrow

Cajal Upgrade

  • went through new version of install.
  • configured session-based remote desktop log in first. This didn’t seem to work.
  • configured virtual host based remote desktop. This seems to give the behavior I want.
  • have to add domain users to permissions setting to allow local log in.
  • installed git-scm under Administrator
    • don’t get Git GUI / Git Bash menus in my personal user account.
    • probably should have added desktop icons, can’t find git-gui.
    • maybe just uninstall and reinstall git tomorrow.

Computer Stuff

  • more useful wmic
    • wmic os get FreePhysicalMemory – return the available RAM
    • wmic cpu get loadpercentage – determine total cpu load percentage
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Monday 04/07/14

9:30a – 11:30p

Meetings

  • lab meeting (see protected notes)
  • my journal club presentation (see google-docs presentation)
  • initial planning meeting with Steven — need joint write-up, goal to write by Wed

Writing

  • finalizing write-up 1 for XZ with Jeff.
  • working on rewriting write-up 2 with team.

Chromatin imaging

  • samples incubated an extra 24 hours.
  • no staining! (in either 647 or 750)
  • hypotheses:
    1. maybe the humidity chamber condensation which hydrates the rubber cement dilutes away the probe?
    • try dry hybe comparison tonight
      1. maybe something is really wrong with new hybe dilution mix
    • I think this mix worked sorta the first time the 750 failed last week.
  • took bead images (currently on STORM2 hard-drive, need to copy over).
  • imaging sample E02

New Staining

  • F07-P1 F06-P3 4:4. .33 A647 and .66 A750
  • new E04-P1 2.5 uL to .33uL A647
  • new E05-P1 1.5 uL to .33uL A647
  • use old hybe mix (not hybe dilution mix)
  • stain in box without water (see how this goes)

Fly Work

  • flipped fly stocks
  • balancer Esc almost dead
  • Esc/CyO dead, but Esc/Gla still going strong (if stable?)
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