Noi/NOTES/2014-6-20

From ZhangLabWiki
Revision as of 01:06, 24 June 2014 by >Noi
Jump to navigation Jump to search

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

PC-P samples

Sample Index
PC_P_1 1
PC_P_2 2
PC_P_3 4
PC_P_4 5
PC_P_5 6
PC_P_6 7
PC_P_7 8
PC_P_8 10
PC_P_9 11
PC_P_10 12
NTC_1 11
NTC_2 12

NC-P samples

Sample Index
NC-1 13
NC-2 14
NC-3 15
NC-5 16
NC-6 27
NC-7 18
NC-8 19
NC-9 20
NC-10 21
NC-11 22
NTC_1 21
NTC_2 22