Noi/NOTES/2014-6-16

From ZhangLabWiki
Revision as of 02:21, 17 June 2014 by >Alan6017518 (→‎Ligation reaction Mix)
Jump to navigation Jump to search

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)