Noi/NOTES/2014-6-28

From ZhangLabWiki
Revision as of 18:14, 28 June 2014 by >Noi (Created page with "= Preparation of RRBS sequencing libraries of solid tumor tissue sample from colon and lung cancer patients = '''Link to calendar''' ==== Sample...")
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to navigation Jump to search

Preparation of RRBS sequencing libraries of solid tumor tissue sample from colon and lung cancer patients

Link to calendar

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!)