Ho Suk:cDNA Rolony construction

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In Situ Whole Transcriptome Amplification From the Cell Monolayer[edit]

  1. Add 10% formalin in PBS for 15 min at RT.
  2. Incubate in 0.25% Triton-X100 in DEPC-PBS for 15 min, OR wash with ethanol for 1 min.
  3. Add 0.1N HCl for 10 min.
  4. Prepare the reverse transcription mixture on ice (important).
    1. 159 ul DEPC-H2O (Ambion)
    2. 20 ul 10x MMLV reaction buffer
    3. 2 ul 25 mM dNTP (Enzymatics)
    4. 2 ul 4 mM aminoallyl dUTP (Anaspec)
    5. 5 ul 100 uM /5Phos/TCTCGGGAACGCTGAAGANNNNNN
    6. 2 ul RNase Inhibitor (Enzymatics)
    7. 10 ul MMuLV reverse transcriptase (Enzymatics)
      • 200ul Total Volume
  5. Add the enzyme cocktail to the fixed cells.
  6. Incubate at RT for 10 min, then transfer to 37°C for overnight.(Day 1)
  7. Aspirate.
  8. Add 20 ul 250 mM BS(PEG)9 in 1 ml PBS for 1 hr at RT.
  9. Block with 1M Tris pH 8.0 for 30 min.
  10. Add 10 ul Riboshredder (Epicentre) in T.E. for 1 hr at 37C.
  11. Rinse the entire well with ddH2O twice to remove traces of phosphate.
  12. Prepare the CircLigase reaction mixture.
    1. 60 ul ddH2O,
    2. 10 ul 10x CircLigase II reaction buffer,
    3. 5 ul 50 mM MnCl2,
    4. 20 ul 5 M Betaine,
    5. 5 ul CircLigase II enzyme
      • 100 ul Total volume
  13. Place in a larger dish with wet towel and incubate at 60C for 2 hr.
  14. Aspirate the reaction mixture.
  15. Prepare the rolling circle amplification reaction mixtures on ice.
    1. 165 ul ddH2O,
    2. 20 ul 10x Phi29 buffer,
    3. 2 ul 25 mM dNTP,
    4. 2 ul 4 mM aminoallyl dUTP
    5. 1 ul 100 uM RCA primer (TCTTCAGCGTTCCCGA*G*A)
    6. 10 ul Phi29 DNA polymerase
      • 200ul Total Volume
  16. Add to cells/tissue and incubate overnight at 30C.
  17. Wash with PBS once.
  18. Add 20 ul 250 mM BS(PEG)9 in 1 ml PBS for 1 hr at RT.
  19. Aspirate well and add 1M Tris pH 8.0 for 30 min.
  20. Store in T.E.


In Situ Whole Transcriptome Amplification From the Fixed Tissue Sections[edit]

  1. Wash with DEPC-PBS.
  2. Incubate with 0.01% Pepsin (Roche) in 0.1 HCl for 30 min.
  3. Prepare the reverse transcription mixture on ice.
    1. 159 ul DEPC-H2O (Ambion)
    2. 20 ul 10x MMLV reaction buffer
    3. 2 ul 25 mM dNTP (Enzymatics)
    4. 2 ul 4 mM aminoallyl dUTP (Anaspec)
    5. 5 ul 100 uM /5Phos/TCTCGGGAACGCTGAAGANNNNNN
    6. 2 ul RNase Inhibitor (Enzymatics)
    7. 10 ul MMLuV reverse transcriptase (Enzymatics)
      • 200 ul Total volume
  4. Add the enzyme cocktail to the fixed cells.
  5. Incubate at RT for 10 min, then transfer to 37°C for overnight.(Day 1)
  6. Aspirate.
  7. Add 20 ul 250 mM BS(PEG)9 in 1 ml PBS for 1 hr at RT.
  8. Block with 1M Tris pH 8.0 for 30 min.
  9. Add 10 ul DNase-free RNases (Roche) in 200 ul 1x NEB#2 for 1 hr at 37C.
  10. Prepare the CircLigase reaction mixture.
    1. 60 ul ddH2O,
    2. 10 ul 10x CircLigase II reaction buffer,
    3. 5 ul 50 mM MnCl2,
    4. 20 ul 5 M Betaine,
    5. 5 ul CircLigase II enzyme
      • 100 ul Total volume
  11. Cover with 2 ml mineral oil and incubate at 60°C overnight.(Day 2)
  12. Aspirate the layer of mineral oil and the reaction mixture.
  13. Prepare the rolling circle amplification reaction mixtures.
    1. 165 ul ddH2O,
    2. 20 ul 10x Phi29 buffer,
    3. 2 ul 25 mM dNTP,
    4. 2 ul 4 mM aminoallyl dUTP,
    5. 1 ul 100 uM RCA primer (TCTTCAGCGTTCCCGA*G*A),
    6. 10 ul Phi29 DNA polymerase
      • 200ul Total Volume
  14. Add to cells/tissue and incubate overnight at 30C.(Day 3)
  15. Wash with PBS on
  16. Add 20 ul 250 mM BS(PEG)9 in 1 ml PBS for 1 hr at RT.
  17. Aspirate well and add 1M Tris pH 8.0 for 30 min.
  18. Store in T.E.