AlanFung:LabNotes/CTCF/2011-7-28

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Revision as of 16:50, 28 July 2011 by >Alan6017518 (Created page with '=Low-input Bisulfite Conversion= * I will be working on the Tagmentation first protocol. ==Protocols Today== * Start with four tubes: 1571 cells, 314 cells, 157 cells, and NTC ==…')
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Low-input Bisulfite Conversion

  • I will be working on the Tagmentation first protocol.

Protocols Today

  • Start with four tubes: 1571 cells, 314 cells, 157 cells, and NTC

Cell Lysis

  • First, make protease Dilution
    • Add 3 uL Protease to 97 uL H2O for a 3:100 dilution
    • Add 1 uL Protease to 99 uL H2O for a 1:100 dilution
  • Next, add 1 uL 3:100 diluted Protease and 1 uL Buffer to 1 uL Frozen cells; incubate at 37 C for 30 minutes, followed by 70 C for 20 minutes
  • Cells are now lysed

Bisulfite Conversion - Imprint Kit

  • Prepare DNA Modification Solution (CT Conversion Reagent):
  • Add 16 uL 0.5 mg/mL BSA and 1 uL Imprint Balance solution to each sample; incubate at 37 C for 10 minutes
  • Add 125 uL DNA Modification solution to each tube, incubate at 65 C for 90 minutes
  • Perform Column Purification of each sample:
    • XXX
    • Elute into 10 uL Elution Buffer

MDA

Tagmentation

  • To lysate, add 1 uL 1:50 diluted Nextera Transposase and 1 uL 5x Nextera HMW Buffer; incubate at 55 C for 5 minutes
  • Next, add 1 uL 1:100 diluted Protease to solution; incubate at 37 C for 30 minutes, followed by 70 C for 20 minutes
  • To this volume, add 1 uL 10 U/uL Klenow exo- Polymerase and 1 uL of methylated dNTPs (1 mM); incubate at 30 C for 30 minutes
  • Library is now ready for Bisulfite Conversion

Amplification

  • Raise volume of any non-bisulfite converted samples to 10 uL (add 2 uL H2O)
  • Add 1 uL Orange Primer, 1 uL Blue Primer, 25 uL KAPA SYBR Supermix, 1 uL BST Polymerase or Klenow Polymerase, and 12 uL H2O to each sample
  • Perform qPCR using the following reaction conditions:
    • 60 C, 20 min incubation (BST second strand synthesis)
    • 72 C, 3 min incubation (Nextera extension (where necessary; not needed if using Klenow))
    • 95 C, 30 seconds (Denature)
    • 35 cycles of:
      • 95 C, 10 seconds (Denature)
      • 58 C, 30 seconds (Anneal)
      • 72 C, 3 minutes (Extend)
    • 72 C, 5 minutes (final extension)
    • Hold at 4 C

Sequencing

  • Hopefully, libraries will pop up as expected.
  • Should probably try some "no-bisulfite" controls...
  • Would need to realiquot cells today; six additional aliquots
  • Talk to Alan.