Alistair's Lab Notebook

9:30 am – 6:20 pm

MERFISH

• finalizing presentation of comparison of pipeline5 and pipeline4
• L8 comparison failed to save before I excited to allow JM sufficient RAM to run. Should have checked first!

RNAi

• primers shipped today, should be here.
• set up PCR reactions
• Phusion calculator for new primers recommends 64C annealing
• 94C melt 3 min start
• 94C 30s melt, 62 C anneal, 72C extend, 34 cycles
• 72 elongate 10 min, 4C hold

Dilutions

• primers, diluted to 100 uM, run at 500 nM (1:20 dilution, 2.5 uL in a 50 uL reaction).
• 1:20 dilution of yw genomic DNA. Use 1 uL
• current dilution of Kc DNA – lets spec
• 2x Phusion HF master mix.

Run

• sample order: Kc-DNA: Th, Rho, Pc-v2, Psc, YW-DNA: Th, Rho, Pc-v2, Psc

Gel 1

• Gene Express ruler (bottom 3 bands are 500, 300 and 100).
  

More templates!

• row order: (Kc-DNA): Su(z)12, SCE, Ph-p, Ph-D, (YW-DNA): ESC, Su(Z)2, SCM, Pho

Cell lines

• on treatment of Su(z)^1b.8 cell line from Schwartz lab
• “We grow them on Schneider’s media from Lonza + 10% FBS. You need to use trypsin to passage the cells, much the same way as it is done for mammalian adherent cultures. And do not dilute the cultures more than to 20% confluence.”

Meetings

• registered for SDB meeting (on Pcard)
• MERFISH lunch team meeting
• discussion with Sharmistha (RK lab) on image processing

10:30 am – 7:00 pm

Meetings

• University IP lawyer (12pm)
• discuss oligopaints with JG

Writing

• started revising DR progress report
• let’s postpone this till after IP discussions
• start work on DF application (not begun)

Presentations

• start making slides for ASBMB meeting next week!

Embryo prep

• flip fly cage, 10:30 am

Probe Design

• design and order Biosearch primers for Hb.
• order placed

Analysis

• comparing Pipeline4 vs Pipeline5
• see post

10:00 am – 5:00 pm

Referee (10:00 am – 1:30 pm)

• finished reading article
• finished writing comments
• summarized comments in review, summarized article contributions
• submitted to editorial office.

Prep for RNAi experiments

Primers

• look up primers on genomernai.org/v14/
• order primers with t7 from IDT

Genomic DNA from Kc cells

• protocol from Filion lab (copied from here)
• Thanks Guillaume!

Material:

• Extraction buffer (see below)
• Proteinase K, 20 mg/mL stock (New England Biolabs, REF: P8102S)
• RNase A, 1 mg/mL stock (Sigma-Aldrich, REF: R6513)
• Chloroform
• Isopropanol
• 70% ethanol

extraction buffer with the following composition.

• 0.8 M guanidine thiocyanate
• 5 mM CaCl2
• 20 mM EDTA
• 5% Tween 20
• 0.5% Triton X-100
• 50 mM HEPES pH 5.3

Procedure:

1. Grow Kc cells in 1.5 mL in a 6-well plate to ∼ 1-2 million cells.
2. Transfer 1.5mL Kc cells into a 1.5 mL Eppendorf.
3. Centrifuge at 2000 rpm for 3 minutes.
4. Resuspend cell pellet with 500 μL extraction buffer.
5. Add 10 μL of proteinase K and 5 μL RNase A. Invert the tube 3-5 times. Do not vortex.
6. Incubate the sample at 55°C for 15 minutes.
7. Add 600 μL chloroform. Emulsify by shaking. Do not vortex.
8. Centrifuge at maximum speed at 4°C for 5 minutes.
9. Transfer the top phase into a new 1.5 mL Eppendorf.
10. Add 350 μL iso-propanol. Invert the tube 3-5 times. Do not vortex.
11. Centrifuge at maximum speed at 4°C for 5 minutes.
12. Spill the supernatant and add 70% ethanol. Invert the tube 3-5 times. Do not vortex.
13. Centrifuge at maximum speed at 4°C for 5 minutes.
14. Spill the supernatant. Do not let dry more than 1 minute.
15. Dissolve DNA pellet in appropriate volume of distilled water.

Alternate

• don’t have Guanidine thiocyanate in stock
• Try the DNAzol approach
• http://www.lifetechnologies.com/us/en/home/references/protocols/nucleic-acid-purification-and-analysis/dna-extraction-protocols/extraction-of-dna-using-reagent.html#proto
• DNA seems to crash out of solution in the optional centrifugation step. Skip this step, get what seem to be good preps.
• prepped 3 samples in 8 mM NaOH. Will test for PCR.

12:00 pm – 12:45pm, 2:30 pm – 7:00 pm

Animal culture

• flip fly plate, 12:30 pm
• passage cells to 75 mm^2 culture flask. Density looks good.

flyMERFISH

• designing fly transcriptome using Jeff’s new pipeline (as an exploration of new pipeline code).
• MORGAN updated and rebooted itself.

Notes on using Transcriptome class

• class annotations for AddAbundances reports only uses cufflinks .fpkm_tracking files. This can’t be true though since the human transcriptome example gets passed my max abundance list from a structure.
• solved: also says accepts name fpkm pairs. These need to be vectors
• found minor bug: mode thrown on a double instead of rounded data. Fixing this substantially improves the number of sequences found per gene.

12:00am -7:00am, 11:30am-7:00pm,

Manuscript

• finished submission, ~7:00 am. yay!

New material to work with:

Embryos

• flipped fly plate, 12:30 pm.
• plan: 3 hr collection + 2 hr age -> 5:30 pm
• flipped cage again 3:30 pm
• depolymerizing formaldhyde at 70C in incubator (should order more, almost out). Need more heptane too.
• plan: fix embryos
• fixed embryos at 5:30 (2-5 hr collection). Good yield. With red label, marked with 2-5hr yw and 15-06-13 in methanol in -20.

Cells

• started new Kc167 culture going from frozen stock, 1:00 pm
• passaged to remove residual freezing media.
• density is excellent, could move to large plates tonight / tomorrow.

Additional prep

• SN points out embryonic derived PSC Su(Z)2 Pirrotta and Schwartz labs
• see experiment planning post describing plans, reagents and resources.

Ordered

• Heptane
• ultapure formaldehyde
• Sneider’s media (for the RNAi and new cell lines. at least necessary for the serum shock transfection).

MERFISH

• catching up on the recent progress be reading the team lab-notebook entries I missed.

Paperwork

• finish descriptions of patents for DR progress report.
• sent forms for signature to XZ.