AlanFung:LabNotes/CTCF/2011-7-28
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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.