Noi/NOTES/2014-6-28
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Preparation of RRBS sequencing libraries of solid tumor tissue sample from colon and lung cancer patients
Sample info
Project ID | Zhang lab ID | Conc. (ng/ul) |
UCSD-006-1 | 6-T_1 | 43.40 |
UCSD-006-2 | 6-T_2 | 31.60 |
UCSD-006-3 | 6-T_3 | 31.50 |
UCSD-006-4 | 6-T_4 | 41.70 |
UCSD-006-5 | 6-T_5 | 30.50 |
UCSD-007-1 | 7-T_1 | 30.70 |
UCSD-007-2 | 7-T_2 | 44.60 |
UCSD-007-3 | 7-T_3 | 35.90 |
UCSD-007-4 | 7-T_4 | 33.80 |
UCSD-007-5 | 7-T_5 | 20.40 |
- X-Y_Z:
- X = project ID number. 4 = pancreas, 6 = colon, 7 = lung
- Y = sample type. T = tissue, P = plasma, S = serum
- Z = sample ID
1) MspI digestion
- Conditions: Digest 100ng of DNA with 20U of MspI in 30ul reaction at 37C O/N (normally 3h) and heat inactivate at 65C 10min
- For 100ng DNA input, I used 2ul (20U) of MspI.
- I spike in 1% of unmeth lambda DNA (1ng)
- I actually start MspI at late night of 2014-06-27, so I incubated it O/N as it doesn't hurt to incubate DNA with MspI longer than 3h.
- I do two replicates for each samples
MspI digestion set up
1.1) Colon samples, UCSD-006
# | Sample | Conc. (ng/ul) | Volume for 100ng (ul) | H2O to 10ul | MspI Mix | Total | Code |
1 | 6-T_1.1 | 43.40 | 2.30 | 7.70 | 20.00 | 30.00 | 6T_1.1 |
2 | 6-T_1.2 | 43.40 | 2.30 | 7.70 | 20.00 | 30.00 | 6T_1.2 |
3 | 6-T_2.1 | 31.60 | 3.16 | 6.84 | 20.00 | 30.00 | 6T_2.1 |
4 | 6-T_2.2 | 31.60 | 3.16 | 6.84 | 20.00 | 30.00 | 6T_2.2 |
5 | 6-T_3.1 | 31.50 | 3.17 | 6.83 | 20.00 | 30.00 | 6T_3.1 |
6 | 6-T_3.2 | 31.50 | 3.17 | 6.83 | 20.00 | 30.00 | 6T_3.2 |
7 | 6-T_4.1 | 41.70 | 2.40 | 7.60 | 20.00 | 30.00 | 6T_4.1 |
8 | 6-T_4.2 | 41.70 | 2.40 | 7.60 | 20.00 | 30.00 | 6T_4.2 |
9 | 6-T_5.1 | 30.50 | 3.28 | 6.72 | 20.00 | 30.00 | 6T_5.1 |
10 | 6-T_5.2 | 30.50 | 3.28 | 6.72 | 20.00 | 30.00 | 6T_5.2 |
11 | 6-T_NTC | 0.00 | 0.00 | 10.00 | 20.00 | 30.00 | 6T_NTC |
1.2) Lung sample, UCSD-007
# | Sample | Conc. (ng/ul) | Volume for 100ng (ul) | H2O to 10ul | MspI Mix | Total | Code |
1 | 7-T_1.1 | 30.70 | 3.26 | 6.74 | 20.00 | 30.00 | 7T_1.1 |
2 | 7-T_1.2 | 30.70 | 3.26 | 6.74 | 20.00 | 30.00 | 7T_1.2 |
3 | 7-T_2.1 | 44.60 | 2.24 | 7.76 | 20.00 | 30.00 | 7T_2.1 |
4 | 7-T_2.2 | 44.60 | 2.24 | 7.76 | 20.00 | 30.00 | 7T_2.2 |
5 | 7-T_3.1 | 35.90 | 2.79 | 7.21 | 20.00 | 30.00 | 7T_3.1 |
6 | 7-T_3.2 | 35.90 | 2.79 | 7.21 | 20.00 | 30.00 | 7T_3.2 |
7 | 7-T_4.1 | 33.80 | 2.96 | 7.04 | 20.00 | 30.00 | 7T_4.1 |
8 | 7-T_4.2 | 33.80 | 2.96 | 7.04 | 20.00 | 30.00 | 7T_4.2 |
9 | 7-T_5.1 | 20.40 | 4.90 | 5.10 | 20.00 | 30.00 | 7T_5.1 |
10 | 7-T_5.2 | 20.40 | 4.90 | 5.10 | 20.00 | 30.00 | 7T_5.2 |
11 | 7-T_NTC | 0.00 | 0.00 | 10.00 | 20.00 | 30.00 | 7T_NTC |
Msp I reaction Mix
Components | 1x rxn | 24 rxn Mix |
MspI (10U/ul) | 2.00 | 48.00 |
10x Tango Buffer | 3.00 | 72.00 |
1ng/ul unmeth-lambda DNA | 1.00 | 24.00 |
H2O | 14.00 | 336.00 |
Total | 20.00 |
Prep
- Aliquot MspI reaction mix to 8-tube strip. 65ul for tube #1-3, #6-8 and 44ul for tube #4-5
- - Add H2O to each tube
- - Add 100ng of DNA to each tube
- - Add 20ul 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, O/N (~h) --> 65C, 20min --> hold at 4C (set heat lid)
2) End-repair/dA-tailing
Prep
- Mix 26ul of Klenow exo- with 26ul of dA:dC:dG (stock 20mM:2mM:2mM) just right before adding to MspI digested DNA
- Aliquot Klenow, exo- and dA:dC:dG mix to 8-tube strip. 7ul for tube #1-3, #6-8 and 5ul for tube #4-6
- - Add 2ul of Klenow, exo- and dA:dC:dG mix to each tube of MspI digested DNA with multichannel pipette. 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)
- Before set up ligation reaction, purify end-repaired DNA with 2x volume AMPure beads (64ul each) and resuspend the bead with 20ul EB buffer
AMPure bead purification
Prep
- Freshly prepare 10mL of 75% EtOH by mixing 7.5mL of 100% EtOH with 2.5mL of H2O (prepare when the tubes sit on magnet)
- Aliquot 1500ul of AMPure beads in 30mL reservoir tube and leave at RT
- - Add 64ul AMPure bead with multichannel pipette. Mix by pipetting 10x
- - Wait for 30min
- - Transfer to sit on magnet for 5min
- - Wash twice with 180ul freshly prepared 75% EtOH
- - After adding 75% EtOH (1st time), wait for 30sec before pipetting up & down 5x
- - Remove all 75% EtOH
- - Dry the bead for 20min
- - Resuspend with 20 EB Buffer (keep the beads in the tubes!)