Matt:LabNotes/2015-6-19

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Motor Neurons DARTFISH CA12kNov14_suppv2

DARTFISH with suppv2

  • Sample labeled "DARTFISH 6.19.2015"
  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 50nM (labeled supp V4 ALL 5.18.15) - 55ul
    • Ampligase Buffer 10X - 10ul
    • Padlock Probes 438nM - 25ul
      • Heat to 90C first and snap cool on ice block
    • Ampligase 5U/ul - 10ul
  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)
  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 (~14hrs)
  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

Imaging