Noi/NOTES/2014-6-29
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Preparation of RRBS sequencing libraries of plasma DNA from colon and lung cancer patients
Sample info
Project ID | Zhang lab ID | Conc. (ng/ul) |
UCSD-006.1 | 6-P_1 | 0.200 |
UCSD-006.2 | 6-P_2 | 1.100 |
UCSD-006.3 | 6-P_3 | 1.570 |
UCSD-006.4 | 6-P_4 | 0.889 |
UCSD-006.5 | 6-P_5 | 0.487 |
UCSD-006.6 | 6-P_6 | 0.249 |
UCSD-006.7 | 6-P_7 | 0.247 |
UCSD-006.8 | 6-P_8 | 0.226 |
UCSD-006.9 | 6-P_9 | 0.408 |
UCSD-006.10 | 6-P_10 | 0.333 |
Project ID | Zhang lab ID | Conc. (ng/ul) |
UCSD-007.1 | 7-P_1 | 0.692 |
UCSD-007.2 | 7-P_2 | 1.140 |
UCSD-007.3 | 7-P_3 | 0.208 |
UCSD-007.4 | 7-P_4 | 0.202 |
UCSD-007.5 | 7-P_5 | 1.110 |
UCSD-007.6 | 7-P_6 | 0.233 |
UCSD-007.7 | 7-P_7 | 0.229 |
UCSD-007.8 | 7-P_8 | 0.167 |
UCSD-007.9 | 7-P_9 | 0.231 |
UCSD-007.10 | 7-P_10 | 0.203 |
- 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
- Note that the protocol of low-input DNA (1-5ng) will be different from the STD RRBS protocol (100ng).
- Conditions: Digest 1ng of DNA with 10U of MspI in 18ul reaction at 37C O/N (normally 3h) and heat inactivate at 65C 20min
- For 1ng DNA input, I used 1ul (10U) of MspI.
- I spike in 1% of unmeth lambda DNA (10pg)
- I actually start MspI at late night of 2014-06-29, so I incubated it O/N as it doesn't hurt to incubate DNA with MspI longer than 3h.
Msp I Reaction Mix
Components | 1x rxn | 26 rxn Mix |
MspI (10U/ul) | 1.00 | 26.00 |
10x Tango Buffer | 2.00 | 52.00 |
12pg/ul unmeth-lambda DNA | 0.83 | 21.67 |
H2O | 8.17 | 212.33 |
Total | 12.00 |
Prep
- Aliquot 39ul of MspI reaction mix to 8-tube strip.
- - Add H2O to each tube
- - Add 1ng of DNA to each tube
- - Add 12ul 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 (~12.5h) --> 65C, 20min --> hold at 4C (set heat lid)
2) End-repair/dA-tailing
Prep
- Mix 29ul of Klenow exo- with 29ul of dA:dC:dG (stock 20mM:2mM:2mM) just right before adding to MspI digested DNA
- Aliquot 7ul of Klenow, exo- and dA:dC:dG mix to 8-tube strip.
- - 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)
3) Methylated adaptor ligation
- Conditions: Add 1ul of 1:20 diluted TruSeq adaptors (do not know actual concentration of adaptors) in 25ul ligation reaction with 6000U of T4 DNA ligase
Ligation reaction set up
Prep
- Thaw diluted TruSeq adaptor on ice (used the diluted adaptors prepared on 2014-06-20, keep it on ice all the time)
- Prepare ligation reaction mix
Ligation reaction Mix
Components 1x rxn 26x rxn dA-tailed DNA 20.00 0.00 10X Tango buffer 0.50 13.00 HC T4 DNA ligase (30units/ul) 1.00 26.00 10mM ATP 1.25 32.50 H2O 1.25 32.50 Total 24.00
Prep
- Aliquot 13ul of ligation reaction mix to 8-tube strip.
- - Add 1ul of 1:20 diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction
Sample TruSeq Index Sample TruSeq Index 6-P_1 13 7-P_1 1 6-P_2 14 7-P_2 2 6-P_3 15 7-P_3 4 6-P_4 16 7-P_4 5 6-P_5 27 7-P_5 6 6-P_6 18 7-P_6 7 6-P_7 19 7-P_7 8 6-P_8 20 7-P_8 10 6-P_9 21 7-P_9 11 6-P_10 22 7-P_10 12 6-P_NTC 22 7-P_NTC 12
- - Add 4ul of ligation reaction mix with multichannel pipette
- - Spin down the tube
- - Mix by gentle pulse-vortexing on PCR rack 10x
- - Spin down the tube
- - 16C, 20h (no heat lid. Ligation time is flexible. I usually do at least 20h at 16C)
- - Mix by gentle pulse-vortexing on PCR rack 5x
- - Quickly spin down
- - Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)
Bisulfite conversion
- Performed bisulfite conversion using LifeTech MethylCode™ Bisulfite Conversion Kit and did the same procedure following manufacturer's instruction and elute with 38ul Elution buffer.
Prep
- - Prepare 2 tubes of complete CT Conversion Reagent by adding 800ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer
- - Mix well and spin down the tube
- - Put on thermomixer at 37C for 15min, 1,000rpm
- - Solution should be clear
- - Spin down the tube
- - Add 120ul complete CT Converson Reagent to 30ul bead-purified adaptor ligated DNA --> total volume 150ul
- - Mix by pipetting 10X with multi-channel pipette
- - Spin down the tube
- - Incubate following below program
- - 98°C for 10 minutes (DNA denaturation)
- - 64°C for 2.5 hours (Bisulfite conversion)
- - 4°C storage for up to 20 hours or continue to desulfonation
- - Add 600ul Binding Buffer to each column
- - Adjust p200 to 200ul and transfer bisulfite treated DNA to the column
- - Mix by pipetting 5x
- - Spin down column at 14,000 rpm for 30s
- - Discard spnt
- - Add 100ul Wash Buffer (Aliquot Wash Buffer in 5mL tube)
- - Spin down column at 14,000 rpm for 30s (do not discard spnt)
- - Add 200ul Desulfonation Buffer (aliquot Desulfonation Buffer in 2mL or 5mL tube)
- - Spin down column at 14,000 rpm for 30s (do not discard spnt)
- - Add 200ul Wash Buffer
- - Spin down column at 14,000 rpm for 30s
- - Discard spnt
- - Add 200ul Wash Buffer
- - Spin down column at 14,000 rpm for 3min (do not discard spnt)
- - Transfer column to prepared 1.5mL LoBind tube & make sure that column does not touch spnt in the collecting tube before transferring to new tube
- - Add 38ul of Elution Buffer (warm Elution Buffer at 60C for 10min)
- - Wait for 2min
- - Spin down column at 12,000 rpm for 1min
- - Continue to amplification (During elution step, PCR mix should be prepared)