Noi/NOTES/2014-9-8
Jump to navigation
Jump to search
Preparation of WGBS libraries of ctDNA/cfDNA from Illumina (no DNA fragmentation step)
Re-quantification of cfDNA by Qubit dsDNA HS assay
Sample | Conc. By Qubit (ng/ul) | Amount (ng) |
cfDNA | 7.09 | 49.63 |
NORM1-02 P1 | 1.66 | 33.2 |
NORM1-03 P1 | 0.77 | 15.3 |
COLO2-03 | 1.11 | 22.2 |
- One sample from Xuyu (received 2014-08-12). I added more TE buffer and re-quantified concentration (7.09ng/ul). The sample was labeled cdDNA without additional further information.
- Note that Alan has taken 10ng of each sample for WGBS library prep . I have no time to wait until the library prep done, so I perform experiment in parallel using 5ng of input DNA to make sure that I can continue BSPP capture and get the library ready for Illumina by this Friday.
1) End-repair/dA-tailing
- Incubate plasma DNA with 5U Klenow fragment, exo- and dA:dC:dG:dT (final conc. 1:0.1:0.1:0.1mM) in total volume 20ul
Zhang lab ID | Conc. (ng/ul) | Volume for 5ng | Exp ID | H2O | Klenow,exo-:dAdCdG Mix | Total | Exp ID |
cfDNA | 7.09 | 0.71 | cf1 | 9.29 | 10.00 | 20.00 | cf1 |
NORM1-02 P1 | 1.66 | 3.01 | cf2 | 6.99 | 10.00 | 20.00 | cf2 |
NORM1-03 P1 | 0.77 | 6.49 | cf3 | 3.51 | 10.00 | 20.00 | cf3 |
COLO2-03 | 1.11 | 4.50 | cf4 | 5.50 | 10.00 | 20.00 | cf4 |
NTC | 0.00 | 0.00 | NTC | 10.00 | 10.00 | 20.00 | NTC |
Klenow exo- and dA:dG:dC:dT reaction mix
Components | 1x rxn | 5.5x rxn |
10X Tango buffer | 2.00 | 11.00 |
dA:dC:dG:dT (20:2:2:2mM) | 1.00 | 5.50 |
Klenow fragment, exo- (5U/ul) | 1.00 | 5.50 |
H2O | 6.00 | 33.00 |
Total | 10.00 |
- - Add H2O to each tube
- - Add 5ng of DNA to each tube
- - Add 10ul of Klwnow, exo- and dA:dC:dG:dT 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
2) Methylated adaptor ligation
- Add 1ul of 1:20 diluted TruSeq adaptors to each end-repaired DNA in total reaction volume 25ul
Ligation reaction mix
Components | 1x rxn | 5.5x rxn |
dA-tailed DNA | 20.00 | 0.00 |
10X Tango buffer | 0.50 | 2.50 |
HC T4 DNA ligase (30units/ul) | 1.00 | 5.00 |
10mM ATP | 1.25 | 6.25 |
H2O | 1.25 | 6.25 |
Total | 24.00 |