Noi/NOTES/2014-6-16
Preparation of RRBS sequencing libraries of solid tumor tissue sample from pancreatic cancer patients
Alan will perform experiment
Sample info
Project ID | Zhang lab ID | Conc. (ng/ul) |
UCSD-004-04 | PC-T-1 | 82.70 |
UCSD-004-05 | PC-T-2 | 35.80 |
UCSD-004-07 | PC-T-4 | 42.80 |
UCSD-004-09 | PC-T-6 | 15.20 |
UCSD-004-10 | PC-T-7 | 16.30 |
1) MspI digestion
- Conditions: Digest 100ng of DNA with 20U of MspI in 30ul reaction at 37C 3h and heat inactivate at 65C 10min
MspI digestion set up
Strip1 | PC-T-1_r1 | PC-T-1_r2 | PC-T-2_r1 | PC-T-2_r2 | PC-T-4_r1 | PC-T-4_r2 | PC-T-6_r1 | PC-T-6_r2 |
DNA conc. (ng/ul) | 82.70 | 82.70 | 35.80 | 35.80 | 42.80 | 42.80 | 15.20 | 15.20 |
Volume for 100ng (ul) | 1.21 | 1.21 | 2.79 | 2.79 | 2.34 | 2.34 | 6.58 | 6.58 |
H2O (ul) | 12.96 | 12.96 | 11.38 | 11.38 | 11.83 | 11.83 | 7.59 | 7.59 |
10X Tango Buffer | 3.00 | 3.00 | 3.00 | 3.00 | 3.00 | 3.00 | 3.00 | 3.00 |
MspI | 2.00 | 2.00 | 2.00 | 2.00 | 2.00 | 2.00 | 2.00 | 2.00 |
12pg/ul unmeth-lambda DNA | 0.83 | 0.83 | 0.83 | 0.83 | 0.83 | 0.83 | 0.83 | 0.83 |
H2O in MspI Mix | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 |
Total (ul) | 30.00 | 30.00 | 30.00 | 30.00 | 30.00 | 30.00 | 30.00 | 30.00 |
Strip2 | PC-T-7_r1 | PC-T-7_r2 | NTC |
DNA conc. (ng/ul) | 16.30 | 16.30 | 0.00 |
Volume for 100ng (ul) | 6.13 | 6.13 | 0.00 |
H2O (ul) | 8.04 | 8.04 | 14.17 |
10X Tango Buffer | 3.00 | 3.00 | 3.00 |
MspI | 2.00 | 2.00 | 2.00 |
12pg/ul unmeth-lambda DNA | 0.83 | 0.83 | 0.83 |
H2O in MspI Mix | 10.00 | 10.00 | 10.00 |
Total (ul) | 30.00 | 30.00 | 30.00 |
Prep
- Label strip tube for reaction set up & UV
- Aliquot H2O in 1.5mL tube & UV
- UV 1.5mL tube for MspI reaction Mix
- Thaw tumor DNA and 12pg/ul unmeth-lambda DNA
- All buffers in this experiment are aliquoted in 8-tube strip for single-use to avoid contamination
Msp I reaction Mix
Components | 1x rxn | 9.5 rxn Mix |
MspI (10U/ul) | 2.00 | 19.00 |
10x Tango Buffer | 3.00 | 28.50 |
12pg/ul unmeth-lambda DNA | 0.83 | 7.89 |
H2O | 10.00 | 95.00 |
Total | 15.83 |
- - Add H2O to each tube
- - Add 100ng of DNA to each tube
- - Add 15.83ul 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
- Note: To preserve enzyme activity, I only take all enzymes from freezer when needed and put it back right after I'm done. Try not to put it on ice for a long time.
Prep
- Mix 13ul of Klenow exo- with 13ul of dA:dC:dG (stock 20mM:2mM:2mM) just right before adding to MspI digested DNA
- - Add 2ul of Klwnow, exo- and dA:dC:dG 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)
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
- 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 5mL of 75% EtOH by mixing 3.75mL of 100% EtOH with 1.25mL of H2O (prepare when the tubes sit on magnet)
- Aliquot 710ul of AMPure beads in 1.5mL tube and leave at RT
- - Add 64ul AMPure bead. 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!)
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 12rxn Mix dA-tailed DNA 20.00 0.00 10x Ligation Buffer 3.00 36.00 T4 DNA Ligase (6000U/ul) 1.00 12.00 TruSeq Adaptor DNA 1.00 0.00 H2O 5.00 60.00 Total 30.00
Ind | Sample |
1 | PC-T-1_r1 |
2 | PC-T-1_r2 |
4 | PC-T-2_r1 |
5 | PC-T-2_r2 |
6 | PC-T-4_r1 |
7 | PC-T-4_r2 |
8 | PC-T-6_r1 |
12 | PC-T-6_r2 |
14 | PC-T-7_r1 |
15 | PC-T-7_r2 |
15 | NTC |
- - Add 1ul of non-diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction
- - Add 9ul of ligation reaction mix
- - Spin down the tube
- - Mix by gentle pulse-vortexing on PCR rack 10x
- - Spin down the tube
- - 16C, 20h (no heat lid)
- - 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 AMPure beads (60ul each) and resuspend the bead with 31ul EB buffer
AMPure bead purification
Prep
- Freshly prepare 5mL of 75% EtOH by mixing 3.75mL of 100% EtOH with 1.25mL of H2O (prepare when the tubes sit on magnet)
- Aliquot 335ul of AMPure beads and 335ul of 20% PEG 8000/5M NaCl in 1.5mL tube, mix well and leave at RT
- - Add 60ul AMPure bead. 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 and transfer 30ul of adaptor-ligated DNA to new strip tubes (discard the beads)
- - Continue to bisulfite conversion
3) Bisulfite conversion
- Performed bisulfite conversion using the same procedure following manufacturer's instruction [[1]] and elute with 38ul Elution buffer.
Prep
- UV H2O
- Label 1.5mL LoBind tube & UV
- Resuspend CT Conversion Reagent (during drying the beads)
- - For 30ul sample: Add 800ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer
- - Mix well and spin down the tube
- - Put on thermomixer at 37C for 15min, 1000rpm
- - Solution should be clear
- - Spin down the tube
- - Add 120ul complete CT Converson Reagent to 30ul bead-purified adaptor ligated DNA
- - 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
- - Spin down column at 14,000 rpm for 30s
- - Discard spnt
- - Add 100ul Wash Buffer (Aliquot Wash Buffer in 5mL tube)
- - Spin column at 14,000 rpm for 30s (do not discard spnt)
- - Add 20ul 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 tramsfering to new tube
- - Add 38ul of Elution Buffer (warm Elution Buffer at 60C for 10min)
- - Wait for 2min
- - Spin down column at 14,000 rpm for 1min
- - Continue to amplification (During elution step, PCR mix should be prepared)