Noi/NOTES/2014-7-31
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Preparation of WGBS libraries of small fragment DNA in plasma samples (TEST)
- Rationale: Will be added
- Aim: To generate WGBS libraries of ctDNA without fragmentation (either enzymatic or shearing) to exclude all large fragment DNA derived from lysed blood cell in sequencing library.
- I chose two samples of PC-P samples which have total DNA more than 10ng to test the protocol
- PC-P-1 (conc. 0.473 ng/ul, total amount 14.19ng)
- PC-P-2 (conc. 0.852 ng/ul, total amount 25.56ng)
1) End-repair/dA-tailing
- Incubate plasma DNA with 5U Klenow fragment, exo- and dA:dC:dG (final conc. 1:0.1:0.1mM) in total volume 20ul
Sample | Conc. (ng/ul) | Volume for 1ng | H2O | Klenow,exo-:dA:dC:dG Mix | Total |
PC-P-1 | 0.47 | 2.11 | 7.69 | 10.20 | 20.00 |
PC-P-2 | 0.85 | 1.17 | 8.63 | 10.20 | 20.00 |
NTC | 0.00 | 0.00 | 9.80 | 10.20 | 20.00 |
Klenow exo- and dA:dG:dC reaction mix
Components | 1x rxn | 3.50x rxn |
10X Tango buffer | 2.00 | 7.00 |
dA:dC:dG (20:2:2mM) | 1.00 | 3.50 |
Klenow fragment, exo- (5U/ul) | 1.00 | 3.50 |
H2O | 6.20 | 21.70 |
Total | 10.20 |
- - Add H2O to each tube
- - Add 1ng of DNA to each tube
- - Add 10.2ul of Klwnow, exo- and dA:dC:dG reaction mix to each tube of DNA. Pipette few times
- - Spin down the tube
- - Mix by gentle pulse-vortexing on PCR rack 10x
- - Spin down the tube
- - 30C, 20min --> 37C, 20min --> hold at 4C (no heat lid)
- - 75C, 10min --> hold at 4C (heat lid on)
- No AMPure bead purification