Noi/NOTES/2014-6-13: Difference between revisions

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:- I precipitated in 3tubes at -80C O/N and resuspended each tube with 11ul H2O --> total volume 33ul
:- I precipitated in 3tubes at -80C O/N and resuspended each tube with 11ul H2O --> total volume 33ul
:- I took 4ul for PAGE verification.
:- I took 4ul for PAGE verification.
==== PAGE verification ====
[[File:ZhangLab_2 2014-06-24 17hr 06min_RRBS_PAGE-verify.jpg| 400px]]
'''Sequencing library ID: NP-RRBS-CTT-100ng, Jun13, 2014'''<br>
'''Sequencing library ID: NP-RRBS-CTT-100ng, Jun13, 2014'''<br>
- Length: 160-360bp<br>
- Length: 160-360bp<br>
- Conc. by Qubit:  XX ng/ul  or  XX nM
- Conc. by Qubit:  XX ng/ul  or  XX nM

Revision as of 00:28, 25 June 2014

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

Components Conc unit Final conc. unit Volume (ul) 6.5 rxn mix
Bis-cvt DNA 6.00 0.00
10X Reaction buffer 10 X 1 X 2.00 13.00
dNTP mix 10 mM 0.25 mM 0.50 3.25
TruS_F/R 10 uM 0.3 uM 0.60 3.90
PfuTurbo Cx 2.5 Unit/ul 2.5 unit 0.40 2.60
50X SYBG 50 X 0.8 X 0.32 2.08
H2O 10.18 66.17
- Aliquot 14 + 6ul DNA template
- Mix well. Split 2X of 9ul
95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 18 cycles --> 72C for 2min
- Stopped at 15X and 17X
- Added 10ul 2X loading dye to each strip tube & mix well
- Load 10ul PCR/dye mix in 12% gel and run at 250 volts for 40min (running in regular 6% TBE gel at 250 volts, 25 min is okay)
File:ZhangLab 2 2014-06-16 13hr 10min CTT-RRBS-test15x-17X.jpg
- Determine # of cycles based on the intensity of the gel. For CTT-frozen samples, I would stop at 15X . For CTT-FFPE samples which have a faint smear, I would stopped at 17X.
  • Then set up reaction 100ul for each samples with 30ul BIS-DNA and split into 2X of 50ul to avoid bias from amplification (Ref. split 4X of 25ul)
Components Conc unit Final conc. unit Volume (ul) 8.5 rxn mix
Bis-cvt DNA 15.00 0.00
10X Reaction buffer 10 X 1 X 5.00 42.50
dNTP mix 10 mM 0.25 mM 1.25 10.63
TruS_F/R 10 uM 0.3 uM 1.50 12.75
PfuTurbo Cx 2.5 Unit/ul 2.5 unit 1.00 8.50
50X SYBG 50 X 0.8 X 0.80 6.80
H2O 25.45 216.33
- I did not include NTC as it clear in TEST PCR
- 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 18 cycles --> 72C for 2min
- Stopped at 15X for CTT-frozen and 17X for CTT-FFPE
- I took 5ul of PCR product from each tube and mix with 5ul of 2X loading buffer to verify that PCR work (even PCR curve looked fine)
- I pooled the two PCR replicatea (total ~87ul for each samples) and purified with 1x volume AMPure beads and eluted with 50ul EB buffer
- I did PAGE quantification by loading 4ul of bead-purified sequencing libraries
File:ZhangLab 2 2014-06-16 17hr 52min CTT-RRBS 100ng PQ.jpg

PAGE quantification results

Sample TruSeq Index Conc. (ng/ul) Amount in 45ul (ng) Volume for 35ng (ul)
CTT-frozen-100ng_1 Ind_18 4.80 215.85 7.30
CTT-frozen-100ng_2 Ind_19 4.64 208.89 7.54
CTT-FFPE-100ng_1 Ind_20 1.05 47.31 33.29
CTT-FFPE-100ng_2 Ind_21 1.77 79.63 19.78
- I pooled 35ng each library based on PAGE quantification and did size selection in 2 well of 5-well 6% TBE gel
File:ZhangLab 2 2014-06-17 14hr 06min 100ng CTTRRBS PAGE-SS.jpg
- I precipitated in 3tubes at -80C O/N and resuspended each tube with 11ul H2O --> total volume 33ul
- I took 4ul for PAGE verification.

PAGE verification

File:ZhangLab 2 2014-06-24 17hr 06min RRBS PAGE-verify.jpg

Sequencing library ID: NP-RRBS-CTT-100ng, Jun13, 2014
- Length: 160-360bp
- Conc. by Qubit: XX ng/ul or XX nM