Thursday 07/23/15

12:30 pm – 7:00 pm

(sick)

RNAi experiments

Primer issues

  • diagnosing problems with primers for qPCR
  • writing code for primer mapping
  • organizing a better data structure to keep track of all the RNAi targets, their qPCR primer sets, their RNAi primer sets, their accession numbers, etc.
  • my overlap checking clearly did not work correctly. Overlap of qPCR and RNAi primers detected for RNA
  • additionally, several of the 20mer primers have perfect hits elsewhere in the genome. Only one should have a sufficiently close compliment to be able to amplify so it shouldn’t be a problem, but still, would have been good to check sooner
    qPCRandRNAiPrimerLocations
  • note Pc_v1, Esc, Pho, and Su(z)2 qPCR primers all overlap RNAi
  • and apparently the polyA selection for cDNA conversion does not sufficiently screen out the added RNA

RNAi new knockdown

  • 2 6-well wells with Ph-p + Ph-D (30 mg/mL)
  • 4 mocks
  • used cells from SFX, rinsed in PBS and repelleted
  • one 75mm2 flask, could have used a few more cells for a bit better density, but it is okay.
  • incubated 55 min.

Cell culture

  • passaged Psc- cells with Trypsin.
  • washed cells with PBS to remove excess serum (inhibits Trypsin)
  • added 3 mL Trypsin soln (now in 4C).
  • After 20 min, they blow off easily. (After 10 they just start rounding up. Next time try just 10 min)
  • add a bit of media to try and inhibit trypsin and pellet cells
  • wash in 5 mL PBS (resuspend and repellet)
  • resuspend and plate in 75mm flask in fresh media.

Embryo in situs

  • remade hybe buffer, dissolving BSA in H2O first (never went into solution otherwise).
  • hydrating embryos into PBT
  • diluted probes (5 nmol) to ~50 uM in ddH20, 50 probes so individual probes at 1 uM. recommended final concentration at 1 nM per probe. Lets try 2 uL in 20 uL hybe buffer O/N. Thats ~10 nM.
Posted in Summaries | Comments Off on Thursday 07/23/15

Wednesday 07/22/15

9:30 am – 8:45 pm

Psc deletion

  • picked up psc deletion cells from Sonny, along with media
  • (also gifted a second aliquot of the Pc-antibody), now frozen.
  • also gifted ~200 mL of media ready mixed.
  • cells were last plated 7/12 and passaged 1/4. Currently ~60% confluent.
  • should remove with trypsin, need to spin down, wash, spin down again, and resuspend to passage.

RNAi experiments

Imaging

  • washed cells, 40 min at 60C in 2x SSC
  • added 100 nm 540/560 beads at 1:3000 (this density looks great)
  • imaging cells – nice and bright, decent density, good contrast. Hopefully these STORM well.
  • the TIRF on STORM2 is all kinds of weird
  • started new AlistairTemp (2 Tb) for data collection on STORM2 (eSATA is functional)
  • installed gitk on storm2 computer
  • copy paste of storm2_splitdax imagewriters.py into latest storm2 branch appears to work fine.

repeat qPCR

  • try lower concentration of cDNA – 1 uL per reaction
  • primers: Pc, Ph, Abd-B, en, Antp, alpha-tub84, Act5A, gapdh1

embryo FISH

protocol from Little 2013

“Embryos were fixed in5%formaldehyde, 1XPBSfor 20 min, and devitellinated as described by Lecuyer et al. (2008). Fixed embryos were rinsed three times in 1X PBS and washed for 10 min in smFISH wash buffer (4X SSC, 35% formamide, 0.1% Tween 20). Hybridization to probes complementary to the reading frame of hb, Kr, kni,or gt and conjugated to Atto 565 (Sigma-Aldrich; 72464) or Atto 633 (Sigma-Aldrich; 01464) was performed for 16–24 hr at a concentration of about 1nMper probe in hybridization buffer (4X SSC,35%formamide,10% dextran sulfate, 2 mg/ml BSA [NEB; B9001], 0.1 mg/ml salmon sperm DNA [In- vitrogen; 15632-011], and 2 mM ribonucleoside vanadyl complex [NEB; S1402S], 0.1% Tween 20). After two washes of 1 hr in wash buffer, embryos were rinsed twice briefly in 1X PBS, stained with DAPI, and mounted in VECTA- SHIELD (Vector Laboratories; H-1000)”

buffers

  • prehybe buffer: 4X SSC, 35% formamide, 0.1% Tween 20 (50 mL master)
    • 10 mL 20x SSC
    • 17.5 mL formamide
    • 500 uL 10% Tween 20
    • fill to 50 mL with ddH2O
  • hybe dilution buffer: 4X SSC,35%formamide,10% dextran sulfate, 2 mg/ml BSA, 0.1 mg/ml salmon sperm DNA, and 2 mM ribonucleoside vanadyl complex , 0.1% Tween 20 (50 mL master)
    • 10 mL 20x SSC
    • 17.5 mL formamide
    • 10 mL 50% dextran sulfate
    • 100 mg BSA
    • 500 uL of 10 mg/mL sonicated salmon sperm DNA
    • RVC
    • 500 uL 10% Tween 20
  • would have been better to pre-disolve the BSA in 10 mL of ddH2O — it does not want to go into solution afterwards. heating to 37, will rock O/N.

Chromatin paper

  • discuss comments with XZ and BB
  • need action items for XZ asap.
Posted in Summaries | Comments Off on Wednesday 07/22/15

Tuesday 07/21/15

10:15 am – 8:00 pm

RNAi experiments

Quantifying knockdown efficiencies by qPCR

cDNA

  1. PPPES (Pc, Ph-p, ph-d, Esc, Scm) rep 1
  2. PPPES (Pc, Ph-p, ph-d, Esc, Scm) rep 2
  3. Pc v1
  4. Pc v2
  5. Pc, Esc, Scm,
  6. Esc
  7. Suz12
  8. Scm
  9. SCE
  10. mock
  11. PPPES (Pc, Ph-p, ph-d, Esc, Scm) rep 0 (from last week)

genes to test

  1. Pc
  2. Ph-p
  3. Esc
  4. Scm
  5. Suz12
  6. SCE
  7. Abd-B
  8. Antp
  9. Ph-d
  10. Gapdh1
  11. Act5A

Experiment layout

  • Note: don’t have primers for Su(Z)12
  • see qPCR setup google doc
  • 2.5 uL of primer mix
  • 2 uL of cDNA

Results

  • knockdown doesn’t seem to have worked so well this time.
  • maybe some issues with the qPCR, will try to run a few lanes again.
    qPCR_plate_150721
  • Notes
    • col 9 has Psc instead of Scm
    • col 10 has Sce instead of Scm
    • col 12 has ‘Psc, Sce, Ph-D and alpha-tub84a’ instead of ‘Abd-B, Antp, Gapdh1, and Act5c’ respectively

new dsRNA synthesis

  • freeze T7 reactions of original PPES combo. Not sure we’re coming back to this.

STORM

  • cells very sparse.
  • note: the top filter wheel sometimes gets turned. It needs to be in position 1. take off the inspection port. if empty port 3 is visible in the inspection port, the correct port 1 is aligned. The IR focus lock beam bounces off this dichroic. No idea why people swap this out.

New stains

  • prepped whole plate of cells
  • should make initial density lower still — a lot of cells detach in sheets during the LN2 treatment.
  • seems to me the PcG RNAi treated cells detach more than the wt / mock cells do.
  • RNase treated just 2 coverslips. Rest still RNA-intact.
Posted in Summaries | Comments Off on Tuesday 07/21/15

Monday 07/20/15

9:00 am – 8:00 pm

Goals

  • prep new RNAi treated cells for staining
  • Plate, fix and lyse current RNAi treated samples
  • RNA isolation prep
  • cDNA production
  • qPCR reactions (?) [tomorrow]
  • Set up synthesis reactions for new RNAi

RNAi experiments

new dsRNA synthesis

  • ran PCR cleanup on new samples of Pc-v2, Psc-v1, Esc, SCM (2 PCR tubes each / 100 uL total reaction)
  • set up T7 reactions:
    • 20 uL eluted DNA-template
    • 20 uL dNTP buffer mix
    • 4 uL T7
    • 4 uL RNasin
  • running for 16 hrs at 37 C

RNA isolation

  • following Qiagen RNAeasy kit
  • the second round elution does make a difference (1.25 – 2x more yield)
  • a third round elution does not help. temperature of water for elution does not matter (tried 4C and 37C)

sample order

  1. PPPES (Pc, Ph-p, ph-d, Esc, Scm) rep 1
  2. PPPES (Pc, Ph-p, ph-d, Esc, Scm) rep 2
  3. Pc v1
  4. Pc v2
  5. Pc, Esc, Scm,
  6. Esc
  7. Suz12
  8. Scm
  9. SCE
  10. mock
  11. PPPES (Pc, Ph-p, ph-d, Esc, Scm) rep 0 (from last week)

* concentrations in the 300 to 800 ng/uL range

First strand cDNA synthesis

RNA dilutions (master 1 12x)

  • (desired 10 ug of RNA) -> 20 uL RNA (have now ~200 ng/uL instead of 2500 ng/uL)
  • 2 uL oligo-dT primer (24 uL)
  • 2 uL dNTP mix (24 uL)
  • heat to 65C for 5 min, return to ice.

per reaction (master 2, 12x)

  • 4 uL 10x buffer (48 uL)
  • 8 uL MgCl2 (96 uL)
  • 4 uL DTT (48 uL)
  • 2 uL RNase OUT (24 uL)
  • 2 superscript3 (24 uL)

sample order

  1. PPPES (Pc, Ph-p, ph-d, Esc, Scm) rep 1
  2. PPPES (Pc, Ph-p, ph-d, Esc, Scm) rep 2
  3. Pc v1
  4. Pc v2
  5. Pc, Esc, Scm,
  6. Esc
  7. Suz12
  8. Scm
  9. SCE
  10. mock
  11. PPPES (Pc, Ph-p, ph-d, Esc, Scm) rep 0 (from last week)

final step

  • add 2 uL RnaseH to each reaction
  • I’ll set up the qPCR reactions tomorrow.
  • samples in -20C in front part of 8-strip rack.

Cell prep for staining

  • prepping all PPPES slides and wt controls for FISH
  • most of these slides have almost unusably low cell denisty
  • staining slide 1 PPPES (0) for Antp-P1 (2 uL + .8 uL S1) O/N.

Prep new cells

  • RNAi from 4 days ago ready to be fixed.
  • Plated 8 wells of new PPES (top for are PPPES rep-1, next for are biological replicate PPPES rep-2), + 2 WT/mock + 2 PES (Pc, Esc, Scm)

New dsRNA synthesis

  • Template making
  • PCR strip order:
  • Ph-p, Ph-D, Ez-v1, Ez-v2, Esc-v2, Psc-v2, Psc-v3, SCE-v2
  • running O/N

New RNAi knockdown

  • 3 wells of 6-well plate with 30 ng Ph-P and Ph-D
  • 3 wells of mock
  • not 100% sure I added the serum free media for the serum shock… Will see when we check the qPCR I guess.

RNAi planning

  • ph looks good, let’s push ahead with this as a single.
  • discussed doing RNA-DNA doubles. RNA with dig in conventional, antibody stained and post-fixed prior to digestion.
  • will try this soon — could use a P1-dig

Comparison of STORM data with Hi-C

  • some initial explorations
    HiC_comparison_Exponents HiC_comparison_InVsOut_bySpot HiC_comparison_InVsOut HiC_density_1 HiC_comparison_density_v2 HiC_comparison_density_v2bar HiC_comparison_density_v1

New Knockdown data

NewKnockdownStructureData

Posted in Summaries | Comments Off on Monday 07/20/15

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Thursday 07/16/15

9:10 am – 1:05 am

MERFISH data analysis

  • Lots of little coding changes

Changes to LoadMERFISHdax

  • Handling collections of info files
  • cell arrays of structures are hard to index
  • these should just be multi-element structures
  • rewriting all info file stacks to conform.

New analysis

  • running InsightM on \\morgan\MorganData2\MERFISHdata\150715_L16Test4\5uM_restain\ Laplace data
  • running daoSTORM on \\morgan\MorganData2\MERFISHdata\150715_L16Test4\5uM_restain\ ‘Raw’ data (post-bleach subtracted data)

additional descriptions and data

  • see my Evernote notebook entry for today (team shared lab-notebook)

Some additional Coding notes

  • wrote MovieToMlist
  • can get running fitting algorithm directly on an image in matlab (i.e. a 3D matrix)
  • can also pass parameters
  • mList = MovieToMlist(dax(:,:,1),'daostormPars',{'threshold','bkd'},'daostormValues',{'100','0'});

RNAi experiments

RNA cleanup

remove template DNA

  • first, DNase digestion of template following NEB (2x since we doubled the RNA synthesis volume) protocol
  • “To remove template DNA, add 30 μl nuclease-free water to each 20 μl reaction, followed by 2 μl of DNase I (RNase-free), mix and incubate for 15 minutes at 37°C.”

cleanup with Agincourt (Beckmann-Coulter) RNA-clean XP beads

  1. Add 1.8 volumes of beads to sample in 1.7 mL tubes (min sample volume 100 uL + 180 beads)
  2. Mix by pipetting, incubate 5-20 minutes at RT (not on magnet)
  3. Separate on magnet until clear
  4. discard solution
  5. rinse 3x in 70% ethanol (500 uL – 1 mL)
  6. let dry 10 minutes
  7. Elute in >30 uL RNase free water

Quality checks

  • RNA concentrations all in the ~1000 ng/uL – 1500 ng/uL range according to nanodrop
  • running on 5% (2 month expired) TBE Urea PAGE gel
  • also forgot to remove tape. Still getting substantial product trapped in wells. Both RNA and DNA ladder look smeary. (somewhat old DNA ladder, maybe it is also a bit degraded). Still, hard to explain the larger than band smears based on degradation when running a denaturing gel.

new RNAi set up with new dsRNA

  • SCM single
  • SCE single
  • Su(Z)12 single
  • Pc single
  • Esc single
  • Pc, Ph-p, Ph-d, Esc, Scm
  • Pc, Esc, Scm (Forgot to make more Psc RNA!!!)
  • water control

New RNAi primers

  • new Psc (PRC1 + dRAF)
  • new SCE/dRING (PRC1 + dRAF)
  • new Esc (PRC2)
  • new Su(Z)12 (PRC2)
  • E(Z) (PRC2)
  • N55 (PRC2) [no sequences]
  • ordered new primers with T7 to make more RNAi

More dsRNA synthesis

  • PCR order (8 tubes)
  • Pc v2, Pc v2, Psc, Psc, Esc, Esc, Scm, Scm.

Data analysis

  • new images of more BX-C cells don’t look that changed (small offset peak, median is still on the original median). Suspect some effects of not excluding single alleles.

Cell culture

  • my BG3 cells might be contaminated. (cells dying)
    • probably an issue with not filter sterilizing the new culture media with the new insulin?
    • hopefully it’s not an issue with the new FBS, this would affect my RNAi as well
    • maybe I got the insulin concentration wrong?
    • don’t see any signs of growth in the media. I’ll leave it out for a while and see what happens
  • restarted frozen stock and transferred to the original Schneider’s in FBS.
    • so far these cells look happy-ish. We’ll see how they’re doing tomorrow
    • (shouldn’t have killed the stock culture yet, but I guess can always re-order)
  • ordered more insulin (got the recommended human insulin in solution instead of the lyopholized cow insulin). Maybe this is what killed my cells. Hope the new stuff arrives.
Posted in Summaries | Comments Off on Thursday 07/16/15

Wednesday 07/15/15

9:10 am – 7:00 pm, 8:50pm – 1:00 am

MERFISH

  • Analyzing MERFISH data, (9:10 am – 1:00 pm)
  • see post
  • MERFISH meeting (1:00 – 2:30 pm)
  • Jeff’s talk (4:00 pm – 5:15 pm)
  • rerunning analysis with nearest third of pixel alignment for drift (previously just nearest pixel, not good enough?)
    • set to decode on non-upsampled images with lower thresholds. see if we get any real RNAs (I think the massive number of potential blanks still wash out this signal).
    • should be able to run pipleline 2 on this data
  • wrote new approach for L16, based on subtraction. (see notes)

To do

  • check sorting of codebook and that final codes, names, and indices match.

RNAi

  • discuss updates and plan of attack with BB (3:00 pm – 4:00 pm)
  • out of dsRNA for more KD – amplifying more template (3x reaction in 3 strips of 6)
  • probe order for PCR:
    1. Pc1,
    2. Pc2 (currently good),
    3. Ph-p,
    4. Ph-d,
    5. esc
    6. scm
  • ran PCR cleanup (pooled triplicates, eluted in 20 uL ddH2O).
  • setup new T7 reactions (20 uL DNTP buffer, 4 uL T7 mix, 2 uL RNAsin Plus) (10:30 pm)
  • current PPhES KD on day 2
  • analyze data: KD results from last batch
    qpcr_plate_RNAi RNAi_exprChangeVsSuZ2 RNAi_exprChangeVsMock
  • ordered primers to GAPDH1, Act5C, and alphaTub84b from IDT (Chrome refuses to do this, redirect loop, ordered through firefox).
Posted in Summaries | Comments Off on Wednesday 07/15/15

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Tuesday 07/14/15

10:00 am – 8:00 pm

Goals

  • make cDNA from RNAi samples from last week
  • qPCR of RNAi samples from last week
  • set up more RNAi for next week
  • make more dsRNA for RNAi
  • reply to Hao, comments on essay
  • analyze Lib15 + 16 data
  • chromatic corrections for L15 data from last week.

Cell culture

  • heat-inactivate fresh aliquot of FBS: 30 min at 56 C with mixing (timing important)
  • DGRC says can’t use Sneider’s from Sigma (http://www.flyrnai.org/DRSC-PRC.html)
  • Made up new BG3 media: 10 ug/mL insulin + 10% heat-inactivated FBS + Schneider’s (from Life Sciences)
  • set up new RNAi. Out of Psc (and not sure it was working anyway)
  • passaged BG3-cells. Set up large flask to grow enough cells to freeze stocks next week.

First strand cDNA synthesis

RNA dilutions (master 1)

  • (desired 10 ug of RNA) -> 20 uL RNA (have now ~200 ng/uL instead of 2500 ng/uL)
  • 2 uL oligo-dT primer (20 uL)
  • 2 uL dNTP mix (20 uL)
  • heat to 65C for 5 min, return to ice.

per reaction (master 2)

  • 4 uL 10x buffer (40 uL)
  • 8 uL MgCl2 (80 uL)
  • 4 uL DTT (40 uL)
  • 2 uL RNase OUT (20 uL)
  • 2 superscript3 (20 uL)

sample order

  1. mock
  2. PSC
  3. PPES (Pc, Psc, Esc, Scm)
  4. Ph-P + Ph-D
  5. Pho
  6. Su(Z)2
  7. HS (heat shock)
    8 RT (room temp cntrl)

QRTPCR reactions

  • Per well, 2.5 uL of chosen primer mix, 2.5 uL of cDNA
  • Per well master
    • 12.5 uL Hot Start Phusion
    • 1.25 uL Eva Green
    • 6.25 uL water
  • 100x total master
    • 1250 Phusion
    • 125 Eva Green
    • 625 uL water
    • 20 uL each
  • running @ 7:33 PM

MERFISH

Chromatic corrections

  • looks like alignment is already pretty good and that accuracy is limited by spot-fitting (of non-point source RNA foci)
  • see Evernote notebook
Posted in Summaries | Comments Off on Tuesday 07/14/15

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