Noi/NOTES/2014-6-20
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Preparation of low-input (1ng) RRBS sequencing libraries of cell-free DNA (plasma samples) from 10 pancreastic cancer patients and 10 normal control
Sample info
1) MspI digestion
- Note that I had condition of MspI digestion of this experiment different from the low-input experiment (5ng) on 06-03-2014 since I used 2ul of MspI in 18ul reaction. I need to increase volume of of Klenow exo- reaction and ligation to 25 and 30ul, respectively to reduce concentration of glycerol in the reaction as there is no purification steps prior to bisulfite conversion.
- For the future, I do not need to use 2ul of MspI as 1ul showed similar activity to 2ul based on agarose gel analysis. I can use the same condition as experiment on 06-03-2014
- I spike in 1% of unmeth lambda DNA. This mean that I included 10pg of lambda DNA for 1ng input DNA.
MspI digestion set up
1.1) PC-P samples
# | Sample | Original conc. (ng/ul) | Volume for 1ng (ul) | H2O to 10ul | MspI Mix | Total | Code |
1 | PC-P_1 | 0.473 | 2.11 | 7.89 | 8.00 | 18.00 | P_1 |
2 | PC-P_2 | 0.852 | 1.17 | 8.83 | 8.00 | 18.00 | P_2 |
3 | PC-P_3 | 1.360 | 0.74 | 9.26 | 8.00 | 18.00 | P_3 |
4 | PC-P_4 | 0.340 | 2.94 | 7.06 | 8.00 | 18.00 | P_4 |
5 | PC-P_5 | 0.813 | 1.23 | 8.77 | 8.00 | 18.00 | P_5 |
6 | PC-P_6 | 0.767 | 1.30 | 8.70 | 8.00 | 18.00 | P_6 |
7 | PC-P_7 | 0.644 | 1.55 | 8.45 | 8.00 | 18.00 | P_7 |
8 | PC-P_8 | 0.524 | 1.91 | 8.09 | 8.00 | 18.00 | P_8 |
9 | PC-P_9 | 0.238 | 4.20 | 5.80 | 8.00 | 18.00 | P_9 |
10 | PC-P_10 | 0.370 | 2.70 | 7.30 | 8.00 | 18.00 | P_10 |
11 | NTC_1 | 0.000 | 0.00 | 10.00 | 8.00 | 18.00 | P_11 |
12 | NTC_2 | 0.000 | 0.00 | 10.00 | 8.00 | 18.00 | P_12 |
1.2) NC-P samples
# | Sample | Original conc. (ng/ul) | Volume for 1ng (ul) | H2O to 10ul | MspI Mix | Total | Code |
1 | NC-1 | 0.293 | 3.41 | 6.59 | 8.00 | 18.00 | N_1 |
2 | NC-2 | 0.325 | 3.08 | 6.92 | 8.00 | 18.00 | N_2 |
3 | NC-3 | 0.538 | 1.86 | 8.14 | 8.00 | 18.00 | N_3 |
4 | NC-5 | 0.156 | 6.41 | 3.59 | 8.00 | 18.00 | N_5 |
5 | NC-6 | 0.148 | 6.76 | 3.24 | 8.00 | 18.00 | N_6 |
6 | NC-7 | 0.245 | 4.08 | 5.92 | 8.00 | 18.00 | N_7 |
7 | NC-8 | 0.141 | 7.09 | 2.91 | 8.00 | 18.00 | N_8 |
8 | NC-9 | 0.171 | 5.85 | 4.15 | 8.00 | 18.00 | N_9 |
9 | NC-10 | 0.134 | 7.46 | 2.54 | 8.00 | 18.00 | N_10 |
10 | NC-11 | 0.152 | 6.58 | 3.42 | 8.00 | 18.00 | N_11 |
11 | NTC_1 | 0.000 | 0.00 | 10.00 | 8.00 | 18.00 | N_12 |
12 | NTC_2 | 0.000 | 0.00 | 10.00 | 8.00 | 18.00 | N_13 |
MspI Reaction Mix
Components | 1x rxn | 26x rxn |
MspI | 2.00 | 52.00 |
10x Tango Buffer | 2.00 | 52.00 |
12pg/ul unmeth lambda DNA | 0.83 | 21.67 |
H2O | 3.17 | 82.33 |
Total | 8.00 |
- - Add H2O to each tube
- - Add 1ng of DNA to each tube
- - Add 8ul of MspI reaction Mix to each tube and pipette few times
- - Spin down the tubes
- - Mix by gentle pulse-vortexing on PCR rack 10x
- - Spin down the tubes
- - 37C, 3h --> 65C, 20min --> hold at 4C (set heat lid)
2) End-repair/dA-tailing
Prep
- I increased reaction volume to 25ul as reason above
Components | 1x rxn | 28x rxn |
MspI-digested DNA | 18.00 | 0.00 |
10X Tango Buffer | 0.50 | 14.00 |
Klenow, exo- | 1.00 | 28.00 |
dA:dC:dG | 1.00 | 28.00 |
H2O | 4.50 | 126.00 |
Total | 25.00 | 196.00 |
- - Aliquot 24.50 of Klwnow, exo- and dA:dC:dG reaction mix to 8-tube strip
- - Add 7ul of Klwnow, exo- and dA:dC:dG reaction mix to each tube of MspI digested 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