Noi/NOTES/2014-9-8

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Revision as of 17:17, 11 September 2014 by >Noi
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Preparation of WGBS libraries of ctDNA/cfDNA from Illumina (no DNA fragmentation step)

Calendar
Sample info.

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