Revision as of 18:37, 22 June 2014 by >Noi
RRBS library preparation of colon tumor tissue samples from Biochain (STD RRBS, 100ng input DNA)
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- 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 12ul H2O
- - I took 3ul for PAGE verification and 2ul for Qubit ssDNA HS quantification.
- Sequencing library ID: NP-RRBS-CTT-100ng, Jun13, 2014
- Length: 160-360bp
- Conc. by Qubit: XX ng/ul or XX nM