Matt:LabNotes/2015-7-20

From ZhangLabWiki
Jump to navigation Jump to search

Motor Neurons Third Batch[edit]

  • Picked up third batch iPS derived motor neurons from Yeo Lab (Sebastian) fixed on 7/17/2015 at 5pm
    • Fixed in 4% paraformaldehyde RT for 15min
    • Permeabilized/store in 70% EtOH at 4C (~72 hours before these experiments)

DARTFISH CA12kNov14_suppv2[edit]

  • Use 1000X concentration of suppressor oligos to match in vitro experiment
      • e.g. 100nM CA12kNov2014_V4 = 28.5pM per padlock probe, therefore 28.5nM of each suppressor oligo


DARTFISH with suppv2[edit]

  • Sample labeled "DARTFISH 7.20.2015"
  1. Wash the cells with 1ml of PBS (RNase free) 3 times
  2. Prepare RT mixture on ice
    • DEPC-H2O - 157ul
    • M-MuLV RT Buffer 10X - 20ul
    • dNTP 25mM - 2ul
    • aa-dUTP 2mM - 4ul
    • RT primer 100uM - 5ul
      • Nonamer
    • RNase inhibitor 40U/ul - 2ul
    • M-MuLV reverse transcriptase 100U/ul - 10ul
  3. Add RT mixture and incubate for 10min at 4C
  4. Transfer sample to 37C overnight (~18hrs)
  5. Wash with 1X PBS once
  6. Add 300ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS prepared on ice) to sample and incubate 1hr at RT
  7. Wash with 1X PBS twice
  8. Add 200ul 1M Tris (pH 8.0) for 30min at RT
  9. Wash with 1X PBS twice
  10. Add RNase Mix (168ul H2O + 20ul RNase H Buffer + 2ul Riboshredder + 10ul RNase H) and incubate 1hr at 37C
  11. Wash with 2ml H2O twice
  12. Prepare Ampligase mixture on ice
    • Suppressor Oligos 100nM (labeled supp V4 ALL 5.18.15) - 25.9ul
    • Ampligase Buffer 10X - 9.1ul
    • Padlock Probes 239nM - 38ul
      • Heat to 90C first and snap cool on ice block
    • Ampligase 5U/ul - 9.1ul
    • H2O - 7.9ul
    • Total: 91ul
  13. Add Ampligase mix to sample and incubate 3hr at 60C then 25hr at 55C (incubator takes a long time to cool from 60C to 55C with door closed)
    • Incubated long time due to timing of starting RCA
    • Middle of glass looked almost dry but edges were clearly wet
  14. Wash with 1X PBS once
  15. Add 200ul (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
  16. Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
  17. Prepare RCA reaction mix on ice
    • H2O - 172ul
    • Phi29 Buffer 10X - 20ul
    • dNTP 25mM - 2ul
    • aa-dUTP 2mM - 4ul
    • Phi29 DNA polymerase 100U/ul - 2ul
  18. Add RCA mix and incubate at 30C overnight (~16hrs)
  19. Wash with 1X PBS once
  20. Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS prepared on ice) to sample and incubate 1hr at RT
  21. Wash with 1X PBS twice
  22. Add 200ul 1M Tris (pH 8.0) for 30min at RT
  23. Wash with 1X PBS twice
  24. Add 0.5uM FISGA_Adpt (1ul 100uM FISGA_Adpt + 199ul 2XSSC + 30% formamide) pre-heated to 75C and incubate 10min at RT
  25. Wash with 2X SSC twice and add 1ml 2X SSC for imaging

Imaging[edit]

  • Check with Universal Rolony FISGA_Adpt
  • ~30 rolonies per cell
    • Not great, could be better
    • High cell density
  • Pos1

File:MAX 20150723 DARTFISH MN FISGA Pos1.jpg

  • Pos2

File:MAX 20150723 DARTFISH MN FISGA Pos2.jpg

sm RNA FISH[edit]

  • Using KIT-488 CUX2-594
    • Expect to see very few to no signals in FITC channel due to few KIT mRNA
    • If TXRED channel shows CUX2 transcripts then dye:probe=1.3 is not the problem
    • If TXRED channel shows bright regions then probe sequences not the problem
  • Using SNAP25-488 CUX2-647
    • Expect to see many SNAP25 spots in FITC channel
    • Hopefully can also see a couple CUX2 spots in Cy5 channel

Procedure[edit]

  • Sample 1 labeled: "RNAFISH1 7.20.2015"
    • KIT-488 CUX2-594
  • Sample 2 labeled: "RNAFISH2 7.20.2015"
    • SNAP25-488 CUX2-647
  1. Prepare 10ml Wash Buffer and let sit at RT
  2. Thaw 300ul aliquot Hybridization Buffer and warm up to 37C
    • Aliquots made by Dan
  3. Prepare hybridization reaction
    • Hybridization Buffer 100ul
    • Probe 5ul + 5ul
  4. Wash the cells with 1ml of PBS (RNase free) 2 times
  5. Add 2ml Wash Buffer and let sit 10min at RT
  6. Aspirate
  7. Add 110ul hybridization solution to each sample and incubate overnight at 37C (~23hrs)
  8. Prepare 12ml Wash Buffer
  9. Warm up 8ml Wash Buffer and 4ml Wash Buffer with DAPI to 37C
  10. Rinse with 2ml Wash Buffer
  11. Add 2ml Wash Buffer and incubate 30min at 37C
  12. Replace with 2ml Wash Buffer with DAPI and incubate 30min at 30C
  13. Wash with 2X SSC (warmed to 30C) twice
  14. Add 2X SSC

Buffer Prep[edit]

  • Wash Buffer
    • 20X SSC 5mL
    • Formamide 5mL
    • RNase free H2O 40ml
  • Wash Buffer with DAPI
    • Wash Buffer 10ml
    • DAPI (100ug/ml) 50ul
      • Stored in brown opaque tube in 4C
  • Hybridization Buffer
    • RNAse free water 5.3 mL
    • SSC 20X 1 mL
    • Dextran sulfate 2 mL
    • Formamide 1 mL
    • E coli tRNA 500 uL
    • RVC 200 mM (warm to 37) 100 uL
    • BSA 50 mg/mL 40 uL

Imaging[edit]

Zeiss Microscope in Huang Lab with Laser excitation[edit]

  • 7/21/2015
  • With help of Matt Walsh
  • Laser wavelengths:
    • 488
    • 552?
    • 642
  • Exposure time: 100ms
  • Sample 1 - 488 (KIT)
    • Nothing to show, probably because gene expression is too low
  • Sample 1 - 594 (CUX2)
    • See combination of bright regions (guess is due to uncoupled dyes) and very few bright spots

File:20150720 RNAFISH1 594.jpg

  • Sample 2 - 488 (SNAP25)
    • Lots of spots but also high background
      • Hard to say significantly more than what I have seen with CUX2 (though based on DARTFISH and FPKM it should be)

File:20150720 RNAFISH2 488.jpg

  • Sample 2 - 647 (CUX2)
    • Image is very grainy making it hard to tell distinct spots from noise, probably needs better excitation parameters

File:20150720 RNAFISH2 647.jpg

Zeiss Microscope in Huang Lab with Metal-Halide light[edit]

  • 7/22/2015