Matt:LabNotes/2015-6-8

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Revision as of 01:17, 9 June 2015 by >Mzcai (→‎FISSEQ)
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RNA in situ Differentiated Motor Neurons

  • 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

  • Sample: RT 15min
    • Labeled "RNAFISH1 6.8.2015"
  • Sample: 37C 10min
    • Labeled "RNAFISH2 6.8.2015"
  1. Prepare 8ml 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 110ul hybridization solution to each sample and incubate overnight at 37C (7p-)

Buffer Prep

  • Wash Buffer
    • 20X SSC 5mL
    • Formamide 5mL
    • RNase free H2O 40ml
  • 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

FISSEQ

  • 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 (7pm-)

DARTFISH

  • 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 (7pm-)