Alistair's Lab Notebook

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

• protocol

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
  

New Knockdown data

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

• online protocol

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.

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
  
• ordered primers to GAPDH1, Act5C, and alphaTub84b from IDT (Chrome refuses to do this, redirect loop, ordered through firefox).

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

• protocol

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