Matt:LabNotes/2015-6-8

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RNA in situ Differentiated Motor Neurons[edit]

  • Picking up iPS derived motor neurons from Yeo Lab (Sebastian)
    • Fixed in 4% paraformaldehyde (fresh) at 8:30a 6/8/2015
      • 4 at RT for 10min
      • 8 at RT for 15min
      • 8 at 37C for 10min
    • Permeabilized/store in 70% EtOH at -20C for ~5hr
      • After bringing to our lab put in 4C > 3hr

RNA FISH[edit]

  • Sample: RT 15min
    • Labeled "RNAFISH1 6.8.2015"
  • Sample: 37C 10min
    • Labeled "RNAFISH2 6.8.2015"
  1. Prepare 10ml Wash Buffer and let sit at RT
  2. Thaw 500ul aliquot Hybridization Buffer and warm up to 37C
    • Aliquots made by Dan
  3. Prepare hybridization reaction (2X volume)
    • Hybridization Buffer 200ul
    • Probe 10ul of each (488 and 594)
  4. Wash the cells with 1ml of PBS (RNase free) 2 times
  5. Add Wash Buffer and let sit 10min at RT
  6. Aspirate
  7. Add 110ul hybridization solution to each sample and incubate overnight at 37C (~18hrs)
  8. Warm up 6ml Wash Buffer and 2ml Wash Buffer with DAPI to 37C
  9. Rinse with 2ml Wash Buffer
  10. Add 1ml Wash Buffer and incubate 30min at 37C
  11. Replace with 1ml Wash Buffer with DAPI and incubate 30min at 30C
  12. Wash with 2X SSC (warmed to 30C) twice
  13. 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

Results[edit]

  • Tried imaging with confocal using 488 and 552 lasers
    • Only saw autofluorescence from cell, looked really cool to see all the dendrites/axons branching from cells but no spots
  • Merged the different channels together (blue=DAPI,green=GFP,red=TXRED,grey=BF)
    • Green and red overlap everywhere (only yellow can be seen) therefore no specific signal
      • Saved in DARTFISH-RNAFISH folder on Desktop (6/10/2015)
      • Will try higher expressed genes (KIT and CUX2): Try #2

File:20150610 MotorNeur RNAFISH2 Dish5 Pos1 composite.jpg

FISSEQ[edit]

  • Sample: RT 15min
    • Labeled "FISSEQ +ctrl 6.8.2015"
  1. Wash the cells with 1ml of PBS (RNase free) 3 times
  2. Prepare RT mixture on ice
    • DEPC-H2O - 159ul
    • M-MuLV RT Buffer 10X - 20ul
    • dNTP 25mM - 2ul
    • aa-dUTP 4mM - 2ul
    • FISSEQ_RT primer 100uM - 5ul
      • /5Phos/TCTCGGGAACGCTGAAGANNNNNN
    • 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 (~15hrs)
  5. Wash with 1X PBS once
  6. Add 300ul cold BS(PEG)9 (6ul stock BS(PEG)9 + 294ul 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 CircLigase mixture on ice
    • H2O - 128ul
    • CircLigase Buffer 10X - 20ul
    • MnCl2 50mM - 10ul
    • Betaine 5M - 40ul
    • CircLigase II 100U/ul - 2ul
  13. Add CircLigase mix to sample and incubate 2hr at 60C
  14. Wash with 1X PBS twice
  15. Add 200ul (1ul 100uM FISSEQ_RCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 60C
  16. Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
  17. Prepare RCA reaction mix on ice
    • H2O - 174ul
    • Phi29 Buffer 10X - 20ul
    • dNTP 25mM - 2ul
    • aa-dUTP 4mM - 2ul
    • Phi29 DNA polymerase 100U/ul - 2ul
  18. Add RCA mix and incubate at 30C overnight (~16 hrs)
  19. Wash with 1X PBS once
  20. Add 300ul cold BS(PEG)9 (6ul stock BS(PEG)9 + 294ul 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 FISSEQ_Adpt (1ul 100uM FISSEQ_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

Results 6/10/2015[edit]

  • Not many rolonies

Pos1
File:Experiment MotorNeurons FISSEQ Pos1 MIP overlay BF.jpg
Pos2
File:Experiment MotorNeurons FISSEQ Pos2 MIP overlay BF.jpg
Pos3
File:Experiment MotorNeurons FISSEQ Pos3 MIP overlay BF.jpg

DARTFISH[edit]

  • Sample: RT 15min
    • Labeled "DARTFISH1 6.8.2015"
  • Sample: 37C 10min
    • Labeled "DARTFISH2 6.8.2015"
  1. Wash the cells with 1ml of PBS (RNase free) 3 times
  2. Prepare RT mixture on ice
    • DEPC-H2O - 318ul
    • M-MuLV RT Buffer 10X - 40ul
    • dNTP 25mM - 4ul
    • aa-dUTP 4mM - 4ul
    • RT primer 100uM - 10ul
      • Nonamer
    • RNase inhibitor 40U/ul - 4ul
    • M-MuLV reverse transcriptase 100U/ul - 20ul
  3. Add RT mixture and incubate for 10min at 4C
  4. Transfer sample to 37C overnight (~15hrs)
  5. Wash with 1X PBS once
  6. Add 300ul cold BS(PEG)9 (6ul stock BS(PEG)9 + 294ul 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 (2X volume) on ice
    • H2O - 110ul
    • Ampligase Buffer 10X - 20ul
    • Padlock Probes 461nM - 50ul
      • Heat to 90C first and snap cool on ice block
    • Ampligase 5U/ul - 20ul
  13. Add Ampligase mix to sample and incubate 3hr at 60C then 21hr at 55C (incubator takes a long time to cool from 60C to 55C with door closed)
    • 3:20p-3p
  14. Wash with 1X PBS once
  15. Add 200ul (1ul 100uM 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 (2X volume) on ice
    • H2O - 348ul
    • Phi29 Buffer 10X - 40ul
    • dNTP 25mM - 4ul
    • aa-dUTP 4mM - 4ul
    • Phi29 DNA polymerase 100U/ul - 4ul
  18. Add RCA mix and incubate at 30C overnight (~14hrs)
  19. Wash with 1X PBS once
  20. Add 300ul cold BS(PEG)9 (6ul stock BS(PEG)9 + 294ul 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
  26. Strip with 80% formamide pre-heated to 75C and incubate 15min at RT
  27. Wash with 1X PBS twice and store in 2ml PBS

Rolony Check 6/11/2015[edit]

  • Much more rolonies than FISSEQ
  • DARTFISH1

File:20150611 MotorNeur DARTFISH1 Pos1 FISGAadpt MIP overlay BF.jpg

  • DARTFISH2

File:20150611 MotorNeur DARTFISH2 Pos1 FISGAadpt MIP overlay BF.jpg

Decoding Results 6/17/2015[edit]