Matt:LabNotes/2015-7-1

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Motor Neurons DARTFISH CA12kNov14_suppv2[edit]

  • Received Bipolar Neurons (iNGN) from Alex at Harvard
    • Fixed in 4% paraformaldehyde (fresh) 6/30/2015
      • RT for 15min
    • Permeabilized/store in 70% EtOH at 4C (~24 hours before this DARTFISH experiment)

DARTFISH with suppv2[edit]

Sample labeled "DARTFISH 7.1.2015"[edit]

  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
    • 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)
    • Center of glass was dry and there was solution around edges of dish
      • The vortexer was shaking all night in the incubator and the vibration may disturb the solution
  5. Wash with 1X PBS once
  6. Add 200ul 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. Store at 4C

Sample labeled "DARTFISH2 7.2.2015"[edit]

  • Because first sample had dry areas after RT preparing a second sample just in case
  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
    • 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 200ul 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. Store at 4C

Continued protocol with DARTFISH2[edit]

  • Only had enough padlock probes for 1 dish so chose DARTFISH2 7.2.2015
  1. Add RNase Mix (168ul H2O + 20ul RNase H Buffer + 2ul Riboshredder + 10ul RNase H) and incubate 1hr at 37C
  2. Wash with 2ml H2O twice
  3. Prepare Ampligase mixture on ice
    • Suppressor Oligos 100nM (labeled V4 supp v2 100nm each ALL (40+5) 7.6.15) - 25ul
      • 10ul V4 5supp 10uM each + 100ul V4 40supp 1uM each + 890ul H2O = 1000ul 100nM each
    • Ampligase Buffer 10X - 10ul
    • Padlock Probes 227nM - 38ul (should be 44ul for 100nM total)
      • Heat to 90C first and snap cool on ice block (FORGOT TO HEAT)
    • H2O - 17ul
    • Ampligase 5U/ul - 10ul
  4. 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)
    • Middle of glass was dry?
  5. Wash with 1X PBS once
  6. Add 200ul (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
  7. Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
  8. 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
  9. Add RCA mix and incubate at 30C overnight (~18hrs)
  10. Wash with 1X PBS once
  11. Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS prepared on ice) to sample and incubate 1hr at RT
  12. Wash with 1X PBS twice
  13. Add 200ul 1M Tris (pH 8.0) for 30min at RT
  14. Wash with 1X PBS twice and store at 4C

Imaging[edit]