Kun:LabNotes/Haplotyping/2011-5-12

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Methylation haplotyping on small number of cells

Idea

  • Directly lyse a small number of cells, remove all proteins, denature the gDNA, and trap the ssDNA in polyacrylamide gel.
  • Perform bisulfite conversion, followed by limited MDA and tagmentation all directly within the gel.
  • Dissect the gel into a number of small pieces, such that each piece contains less than haploid genome, followed by barcoded PCR in tubes.
  • The PCR amplicons are pooled for sequencing.

Experiment protocol

  • Make dilution of GM20431 cells to 1 cell/ul.
  • Mix 10ul of cell solution with 10ul Cell Lysis Buffer(20M EDTA, 10mM Tris.HCl , 200mM NaCl, 0.2% Triton X-100, 0.2AU/ml Qiagen Protease), incubate at 37C for 30min, 75C for 15min. Slowly mix the cell lysate with a P20 pipette for 10 times (to break down chromosomes).
  • Prepare gel mix:
      H2O	           10ul
      ABD	            8ul
      30% BSA            0.3ul  
      Cell lysate         20ul
      5% TEMED           0.8ul
   Heat at 94C for 5min, immediately transfer to ice
   Add 5% APS 	  0.8ul 
  • Add 18ul each to two slides, wait ~15 minutes for the gel to polymerize.
  • Wash the slides with ddH2O for >5min.
  • Place a frameseal chamber on each slide, add 120 ul CT Conversion Reagent (Zymo EZ DNA Methylation Direct Kit), seal the chamber.
  • Incubate at 95C 4min -> 64C 4h -> 4C hold.
  Roughly half of the CT conversion reagent evaporated in less than 2 hours despite my best efforts in sealing the chamber.
  So I removed the chambers, put new ones and added new CT Conversion Reagent. 
  95C 1min -> 64C 2h 15min -> 4C hold.
  • Wash the slides twice with ddH2O for >5min.
  • Add ~50ul of M-desulphonation buffer onto the gel and let it stand at RT for 15min.
  • Wash the slides twice with ddH2O for >5min.
  • Set up limited MDA reaction:
     10x RepliPhi Buffer       4.0ul
     1mM N6 primer             2.0ul
     25mM dNTP                 0.4ul
     2X SYBR Green I           2.0ul
     RepliPhi Phi29(100U/ul)   2.0ul
     Exo-minus Klenow (5U/ul)  1.0ul
     H2O                      28.0ul
     30% BSA                   0.7ul
   Incubate once slide at 30C for 30 min, the other slide at 30C for 1h, head inactivation at 65C for 10min
  • Wash the slide with ddH2O for > 5min, air-dry inside PCR hood.
  • Prepare tagmentation mix:
   Dilute the enzyme: 1:5     
     5x LMW Buffer             8ul
     diluted enzyme            4ul
     H2O                      28ul
   Add 20ul to each gel, cover with a coverslip, incubate at 55C for 10min
  • Wash the slide with ddH2O for > 5min, use a clean scalpel to cut the gel into 12 slices, transfer one into each PCR tube.
  • Set up PCR reaction:
                                    x 12
     KAPA QPCR mix            25ul
     Orange Primer (10uM)      1ul
     Blue Primer (10uM)        1ul
     Bst Pol (5U/ul)         0.5ul
     H2O                      23ul
     65C 15min -> 95C 30 sec -> (95C 10sec -> 58C 30 sec -> 72 1min) x 30 -> 72C 3min. 
    Monitor the reactions on a real-time thermal cycler and terminate them before the curves reach saturation.
  • If the amplification curves are good, check the amplicon size with 6% TBE gel.
  • If the amplicons have the right size, perform 2nd PCR to add barcodes and sequencing adaptors.