Noi/NOTES/2014-6-13

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RRBS library preparation of colon tumor tissue samples from Biochain (STD RRBS, 100ng input DNA)

  • [Link to calendar]
  • After I have tried STD protocol to get it worked in 12878 sample, I continued to work on CTT samples.
  • The main things to concern are concentration of adaptor concentration in ligation reaction and optimization of PCR cycle number to prevent over-amplification of sequencing libraries.
  • In this experiment, I spiked in 10pg of unmethylated lambda DNA which accounts for 0.01% of input DNA. This might be too low.

Experimental procedures

  • Sample list
    • CTT-frozen, 100ng, two replicates
    • CTT-FFPE, 100ng, two replicates

1) MspI digestion

  • Conditions: Digest 100ng of DNA with 20U of MspI in 30ul reaction at 37C 3h and heat inactivate at 65C 10min
Sample Conc. (ng/ul) Volume for 100ng (ul) 10X Tango Fuffer MspI (10U/ul) H2O to adjust volume (ul) 12pg/ul unmeth-lambda DNA H2O in MspI mix(ul) Total (ul)
1.1 CTT-frozen 52.30 1.91 3.00 2.00 12.26 0.83 10.00 30.00
1.2 CTT-FFPE 64.80 1.54 3.00 2.00 12.63 0.83 10.00 30.00
1.3 NTC 0.00 0.00 3.00 2.00 14.17 0.83 10.00 30.00

Msp I reaction Mix

Components 1 rxn 5.5 rxn mix
MspI (10U/ul) 2.00 11.00
10X Tango Buffer 3.00 16.50
12pg/ul unmeth-lambda DNA 0.83 4.57
H2O 10.00 55.00
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, 12h --> 65C, 20min --> hold at 4C (set heat lid)
- Note: For this experiment, I set up MspI at night, so I incubate O/N. Longer incubation did not increase digestion that much. However, it has been reported that it does not cause star activity.

2) End-repair/dA-tailing

Prep
- Mix 6.5ul of Klenow exo- with 6.5ul 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)
  • 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!)

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 325ul 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 Mix

Compinents 1x rxn 5.2x rxn
dA-tailed DNA 20.00 0.00
10X Ligation Buffer 3.00 15.60
T4 DNA ligase 1.00 5.20
H2O 5.00 26.00
total 29.00
- 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
Sample TruSeq Index
CTT-frozen-100ng_1 Ind_18
CTT-frozen-100ng_2 Ind_19
CTT-FFPE-100ng_1 Ind_20
CTT-FFPE-100ng_2 Ind_21
NTC Ind_22

AMPure bead purification

Prep
- Freshly prepare 4mL of 75% EtOH by mixing 3mL of 100% EtOH with 1mL of H2O (prepare when the tubes sit on magnet)
- Aliquot 155ul of AMPure beads and 155ul 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

4) Bisulfite conversion

  • Performed bisulfite conversion using the same procedure following manufacturer's instruction [[1]] and elute with 38ul Elution buffer.

Prep

- Mix well and spin down the tube
- Put on thermomixer at 37C for 15min, 1000rpm
- Solution should be clear
- Spin down the tube
- Note: I used CT conversion resuspended on 2014-06-11

- 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 transfering 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