Noi/NOTES/2014-6-28
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 20min
- 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 sample
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 (~13.5h) --> 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 8mL of 75% EtOH by mixing 6mL of 100% EtOH with 2mL of H2O (prepare when the tubes sit on magnet)
- Aliquot 1400ul of AMPure beads in 30mL reservoir 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!)
3) Methylated adaptor ligation
- Conditions: Add 1ul of non-diluted TruSeq adaptors (do not know actual concentration of adaptors) in 30ul ligation reaction with 6000U of T4 DNA ligase
Ligation reaction set up
Prep
- Thaw TruSeq adaptor on ice (during drying the beads, keep it on ice all the time)
- Prepare ligation reaction mix (during drying the beads)
Ligation reaction Mix
Components 1x rxn 24rxn Mix dA-tailed DNA 20.00 0.00 10x Ligation Buffer 3.00 72.00 T4 DNA Ligase (6000U/ul) 1.00 24.00 TruSeq Adaptor DNA 1.00 24.00 H2O 5.00 120.00 Total 30.00
Prep
- Aliquot ligation reaction mix to 8-tube strip. 29ul for tube #1-3, #6-8 and 20ul for tube #4-5
- - Add 1ul of non-diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction
Sample TruSeq Index Sample TruSeq Index 6-T_1.1 1 7-T_1.1 13 6-T_1.2 2 7-T_1.2 14 6-T_2.1 23 7-T_2.1 15 6-T_2.2 4 7-T_2.2 16 6-T_3.1 5 7-T_3.1 27 6-T_3.2 6 7-T_3.2 18 6-T_4.1 7 7-T_4.1 19 6-T_4.2 8 7-T_4.2 20 6-T_5.1 25 7-T_5.1 21 6-T_5.2 10 7-T_5.2 22 6-T_NTC 10 7-T_NTC 22
- - Add 9ul 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, 24h (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)
- Before continue to bisulfite conversion, purify adaptor-ligated DNA with 2x volume diluted AMPure beads (60ul each) and resuspend the bead with 31ul EB buffer
2014-06-29
AMPure bead purification
Prep
- Freshly prepare 8mL of 75% EtOH by mixing 6mL of 100% EtOH with 2mL of H2O (prepare when the tubes sit on magnet)
- Aliquot 670ul of AMPure beads and 670ul of 20% PEG 8000/5M NaCl in 1.5mL tube, mix well and leave at RT (forgot to dilute, used beads directly)
- - Add 60ul 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 31 EB Buffer.
- - Wait for 2min.
- - Transfer to sit on magnet for 2 min and transfer 30ul of adaptor-ligated DNA to new strip tubes for bisulfite conversion (discard the beads)
- - Continue to bisulfite conversion
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)
Amplification
TEST PCR
Components | Conc | unit | Final conc./amount | unit | Volume (ul) | 25 rxn mix |
Bis-cvt DNA | 6.00 | 0.00 | ||||
10X Reaction buffer | 10 | X | 1 | X | 2.00 | 50.00 |
dNTP mix | 10 | mM | 0.25 | mM | 0.50 | 12.50 |
TruS_F/R | 10 | uM | 0.3 | uM | 0.60 | 15.00 |
50X SYBG | 50 | X | 0.8 | X | 0.32 | 8.00 |
PfuTurbo Cx | 2.5 | Unit/ul | 1 | unit | 0.40 | 10.00 |
H2O | 10.18 | 254.50 | ||||
Total | 20.00 |
- - Aliquot 14 + 6ul DNA template
- - Mix well. Split 2X of 8.5ul
- 95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X [14X & 16X] --> 72C for 2min
- There was error to realplex during annealing step of 15 cycles. I needed to remove samples. I loaded only 14X to save the gel and plan to loaded 16 cycles PCR if the intensity of 14X is too low.
- - Add 10ul of 2X loading dye to PCR product and load 10ul PCR product/dyemix in 12% gel and run at 250 volts for 40min
Colon tumor samples File:ZhangLab 2 2014-06-30 17hr 16min 6T 100ngRRBS TEST-14X.jpg Lung tumor samples File:ZhangLab 2 2014-06-30 17hr 16min 7T 100ngRRBS TEST-14X-e.jpgg
- I do not know why sample 7-T_3.2 and 7-T_4.1 failed. May need to check with Dr. Zhang if he want to sequence the two duplicates or only 1 library for each sample.
- No need to load 16 cycles PCR since 14 cycles look good and not too faint or over-amplified.
- I need to repeat 7-T_3.2 and 7-T_4.1. I plan to repeat both of them in two duplicates.
- Continued to prepare sequencing library by doing PCR in 100ul reaction with 30% BIS-DNA.
Components | Conc | unit | Final conc./amount | unit | Volume (ul) | 20.5 |
Bis-cvt DNA | 30.00 | 0.00 | ||||
10X Reaction buffer | 10 | X | 1 | X | 10.00 | 205.00 |
dNTP mix | 10 | mM | 0.25 | mM | 2.50 | 51.25 |
TruS_F/R | 10 | uM | 0.3 | uM | 3.00 | 61.50 |
50X SYBG | 50 | X | 0.8 | X | 1.60 | 32.80 |
PfuTurbo Cx | 2.5 | Unit/ul | 1 | unit | 2.00 | 41.00 |
H2O | 50.90 | 1043.45 | ||||
Total | 100.00 |
- - Aliquot 70 + 30ul DNA template
- - Mix well. Split 2X of 50ul (actual ~48ul)
- 95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 14 cycles --> 72C for 2min
- - Stopped at 14X cycles based on TEST PCR
- - I pooled the two PCR replicates (total ~90ul for each samples) and purified with 1.2x volume AMPure beads and eluted with 55ul EB buffer
- - I did PAGE quantification by loading 3ul of bead-purified sequencing libraries
File:ZhangLab 2 2014-07-02 19hr 34min PQ 6T 100ngRRBS.jpg File:ZhangLab 2 2014-07-02 19hr 34min PQ 7T 100ngRRBS.jpg
- It's so wired for sample 6T_4.2 since it looked fine in TEST PCR, but it's very faint after amplification in 50ul and bead purified.
Repeat smaple 7T_4.1 (also include 7T_4.2) File:ZhangLab 2 2014-07-03 17hr 35min PQ repeat 7T 4.jpg