Kun:LabNotes/Haplotyping/2011-5-12
Jump to navigation
Jump to search
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.
- 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.