4:00 pm

tasks

• send Fano data to ST

L11 analysis

• CreateWords is slow, probably worth writing a faster version of this.
• somehow pipeline2 is reporting 40 cells when there should only be 26, not sure what happened here — troubleshooting.
• between Jeff’s analysis and running pipeline2 on the L11 data Morgan crashed.

9:00 am – 7:30 pm, 9:00 pm – 11:30 pm

Thesis defense

• finalizing hat for Hao 9am-10am
• Hao’s Thesis defense 10am-11:15 am
• more finalizing hat 11:15-11:45

Discussions 11:45-1:00pm

• discussion of AO with HB and BB
• discussion of BB’s sequential stain data — look’s awesome!

Presentation Prep

• working on slides for CSHL meeting
• working on slides for Ph-polymerization project summary for tomorrow lab meeting

MERFISH

Developing New Pipeline for Multicolor data 7:00pm – 11:20 pm

• attempting pre-warp
• using brightfield bead movies and CorrAlign
• adjusted CorrAlign to dilate images before computing correlation
  • this is computationally expensive. 5x is quite slow. 3x is okay.
  • this does provide sub-pixel improvement
• TranslateImage however is pixel based, doesn’t provide sub-pixel translation.
  • could make sub-pixel version — just dilate, then translate.

Questions for team

• why 3 color 8 hybes for L7 (only 16 bits not 24…)
• there are clearly focus lock issues still…
• why is 10 uM scale of primer $400!! (it would be cheaper to order 10x 1uM scale, ~$300)

Ph Project

• reply to NJ about abstract
• to do tomorrow: rewrite model discussion section
• re-arrange the rest of the Fig 5 panels

9:00 am – 5:00 pm, 7:45 pm – 10:45 pm

practice talk (9am-10am)

Lab meeting 10am – 1:30pm

• see protected notes

Embryo MERFISH project

Embryo staining

STORM3 observations of cut sections

• some individual spots look promising.
• despite high concentration of probe, background does not look any higher / not too bad (still pretty clear to see tissue, no mistaking cytoplasm with slide background. Cytoplasm background substantially higher than nuclear background.

confocal observations

• some staining of tsl, not substantially better than samples from yesterday stained at lower concentration in 35% formamide.
• sample quality I think is compromised — some nuclear morphology looks poor, DAPI channel background in the cytoplasm looks unexpectedly high, and nuclei are not as bright as I would expect for a substantial, long incubation in more concentrated DAPI (1:100 dilution instead of 1:5000 of what I believe is a 5 mg/mL stock).
• conclusion: we can get some staining. Further diagnosis will be safer with fresher samples. Let’s cut fresh embryos. Compromised sample integrity may have been a problem in previous chromatin stains as well.

Fly work

• transferred both wildtype tube adults to new bottles. By the time I get back from Genome Biology meeting there should be plenty of these.
• if GS joins the lab we can start working from new embryos together.

9:00 am – 10:15 pm

Microscope booking

• booked STORM3 for imaging this evening
• STORM2 in use, Andor camera moved to STORM5 construction scope
• STORM4 may be available for checking samples
• re-applied for expense code for confocals.
• registered for 7pm – 8pm to for confocal LSM 700 to check embryos

Embryo staining

• embryo sections on coverglass on elevated platforms dried out :(. Should seal with rubber-cement or cover with a wet kimwipe. humidity chamber not working too well.
• no staining detected in Tll sections
• check embryos on Turnkey? — No camera
• check embryo staining on LSM 700 — no staining detected in blastoderm embryos. Late embryos seem to have some staining around the CNS and around the

Staining try 2

• 10 uL in 100 uL of hybe dilution buffer made with 10% dextran sulfate and 10% formamide.
• not using spacer on slides this time, 20 uL of staining buffer, sealed with rubber cement.
• added embryos (~15 uL) to remaining 80 uL of hybe dilution buffer with 10 concentrated probe
• (this time its clearly blue, so probe concentration is super high). I expect we get background and I can work from here washing it down. I hope we get decent signal too though. There didn’t even seem to be good single molecule background signal when tested on STORM4 today.

more problems

• something went wrong in probe assembly, probes sequences for tll map to tsl:
• this looks something like the confocal pattern today (From BDGP):
  
• and my ‘Kr’ probe is mapping to lea, (aka robo2), no pattern recorded in BDGP. should have checked these earlier.
• found bug in code: GeneSelection_150325. gene names got mismatched with sequences during select longest isoform phase…
• need more investigation tomorrow…

Oligopaints team meeting

• at Wu lab.
• good presentations,
• hierarchical scales of coding from Sonny look good
• resolution depth and speed from Peng Yin lab look pretty awesome.

9:00 am – 5:30 pm, 9:00 pm – 10:00 pm

Morning tasks

• Finish ultra-cryo cleanup (put knives away)
• Discuss drive issue with Dell.
  • system currently configured with only 4 bays active
  • need RAID controller to connect 8 bays together and pass those to the motherboard
  • this requires reformatting the drives.

In Situs

whole mount embryo in situs

• move from methanol to ethanol (through 50:50)
• move to xylenes (through 50:50)
• treat in xylenes, 1.5 hours
• move to ethanol
• move to PBS
• post fix, 25 min in 5% PFA in 1/2 PBT
• 8 min Sodium borohydride treatment
• rinse in PBT
• Trying Little’s pre-hybe buffer: 4x SSC, 35% formamide, 0.1% Tween-20, 10 min
  • in 100 mL: 20 mL 20x SSC, 35 mL formamide, 1 mL 10% Tween-20. rest H2O
  • in 50 mL: 10 mL 20x SSC, 17.5 mL formamide, 0.5 mL 10% Tween-20, fill to 50 mL
• Trying Little’s hybe buffer: 4x SSC, 35% formamide, 10% dextran sulfate 2ug/mL BSA, 0.1 mg/mL salmon sperm, 2 mM ribonucleoside vanadyl complex, 0.1% Tween-20 + 1 nM per probe.
  • in 10 mL: 2 mL 20x SSC, 3.5 mL formamide,2 mL dextran sulfate, 100 uL 10mg/mL salmon sperm, 100 uL 10% Tween-20.
• probes ship at 5 nmols
• that’s ~50 probes, so dissolving this in 100 uL each probe is at 1 uM.
• so let’s dissolve in 200 uL RNase / DNase free water, then dilute at 1:500 for experiments. — actually, this stock looks much more dilute than I expect, let’s try 2:500 (not that this will make a difference relative to 1:500).

sectioned embryo in situs

• dissolve sucrose and stain with Hoechst (30 min)
• post-fix 15 min in 5% PFA in PBS
• from here same as above after post fix.
• sections clearly visible on coverslip if light catches correctly.
• saved 2 coverslips to repeat at higher concentration if staining fails.

Ordering

• placed orders

Meetings

• discussion with Sonny, GN, BB, and SW on chromatin projects
• MERFISH discussion

9:00 am – 10:50 pm

Computer maintenance

• finish install of updates. Shut-down Morgan
• install power adapter for USB 3.0 cards bridge to DVD drives
• investigate motherboard for ID# (Dell special, no additional info) and RAID card (none evident)
• restart computer
• Morgan still not recognizing added disk drives.
• formatted one of the disks on Monet and remounted in Morgan. Still not recognized
• rebooted Morgan. Drive still not recognized. 🙁
• swapping existing F with new drive (turned off).
• mounted wrong in bracket. Needs to be in SAS, not SATAmu position.
• recognizes top rack of drives. Still not recognizing bottom rack, despite the fact these clearly do plug into the motherboard.

Further investigation of the issues

• http://en.community.dell.com/support-forums/servers/f/906/t/19568567
• try customer service chat tomorrow morning

Embryo work

• polyD lysine coated second rack of coverslips (others are 5x chromium gelatin)
• polyD lysine coverslips easily ID’d from salt residue
• sectioned embryos, used all coverslips from both rack
• cut until validated that sections contain embryos.
• mostly cut at at 2 um, -62C blade and mount, -55C chamber. Sections coming very nicely off blade today, limited coiling.
• ordered new loops for ultra-cryo section collection
• froze along with the pin

MERFISH

• Processing probes in 15-04-19_Probes_18mers_t66Cx76Cs99C
• 18 – 22mers.
• 186 genes in my list have non-unique common names / gene symbols
• 5,015 have FPKM > 0.1 (max over cell lines) and more than 100 probes.
• Maybe 76C is too strict for off target homology cut.

Human transcriptome rebuild

• Currently building annotation off human chromosome fasta files.
• sent new transcriptome fasta file to Jeff for all protein coding genes

10:00 am – 5:00 pm

Meetings

• discussion with NL about MERFISH 10 am – 11 am
• reply to E Furlong about MERFISH

BWF prep

• call foundation about slide submission
• rehearse presentation timing
• feedback from Jeff on slides
• feedback from Bogdan on slides
• sent slides (2 versions, with and without animation appearances)
• sounds like animation version is okay.

April planning

• Need slides for CSHL Genome Biology meeting
• schedule practice talk for CSHL Genome Biology meeting

To order

• 18 mm round coverglass, #1.5. 72222-01 from Electron Microscopy Sciences
• forceps kit (my forceps keep getting stolen)

5:00 pm – 12:00 am

MERFISH

• Transcriptome building
• saving additional FPKM data per cell line, per chosen isoform,
• rebuilding FASTA file with short gene names.
• running with maxFPKMperType > 1E-5 & maxFPKMperType < 50E3 and fasta length over 2000 bp (12024 genes).

To do

• order primers for L12
• design L13

BWF

• finalize slides!!