Noi/NOTES/2014-6-16

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Preparation of RRBS sequencing libraries of solid tumor tissue sample from pancreatic cancer patients

Alan will perform this experiment

Sample info

Noi/NOTES/2014-6-9

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 11.5 rxn Mix
MspI (10U/ul) 2.00 23.00
10x Tango Buffer 3.00 34.50
12pg/ul unmeth-lambda DNA 0.83 9.55
H2O 10.00 115.00
Total 15.83
  • I should have prepared 11.5x rxn -> fixed
- 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)
  • 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

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 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 (forgot to dilute, used beads directly)

- 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 21 EB Buffer.
- Wait for 2min.
- Transfer to sit on magnet for 2 min and transfer 20ul of adaptor-ligated DNA to new strip tubes (discard the beads)
- Continue to bisulfite conversion
- Note: To save time, I told Alan to switch to use Zymo EZ DNA Methylation-Lightning™ Kit since it save time and we can resuspend adaptor-ligated DNA in smaller volume (21ul)


4) Bisulfite conversion

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

Prep
- UV H2O
- Label 1.5mL LoBind tube & UV

- Add 130ul complete Lightning Converson Reagent to 20ul bead-purified adaptor ligated DNA --> total volume 150ul
- Mix by pipetting 10X with multi-channel pipette
- Spin down the tube
- Incubate following below program (Magprep, Heated Lid)
- 98°C for 8 minutes (DNA denaturation)
- 54°C for 1 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)
- Wait for 18min
- 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

  • Alan has gone through bisulfite conversion, and I continued to do amplification.
  • To monitor the exact number of PCR cycles,
    • Set up PCR reaction volume 20ul with 6ul DNA template in strip tube.
    • Mix well and split into two strip tubes (9ul each)
    • Run PCR and stopped reaction at 15X and 17X

TEST PCR

Components Conc unit Final conc./amount unit Volume (ul) 13 rxn mix
Bis-cvt DNA 6.00 0.00
10X Reaction buffer 10 X 1 X 2.00 26.00
dNTP mix 10 mM 0.25 mM 0.50 6.50
TruS_F/R 10 uM 0.3 uM 0.60 7.80
50X SYBG 50 X 0.8 X 0.32 4.16
PfuTurbo Cx 2.5 Unit/ul 1 unit 0.40 5.20
H2O 10.18 132.34
Total 20.00
- 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 (equivalent to 5ul PCR product) in 12% gel and run at 250 volts for 40min (running in regular 6% TBE gel at 250 volts, 25 min is okay)
- Determine # of cycles based on the intensity of the gel. If the smear between 160bp -360bp is too bright and has some background a long the lane, we can reduce number of cycles ~1-2x. If the smear is very faint, increase number of cycle ~1. Based on CTT-RRBS 100ng input experiment, I would allow PCR between 15-18 cycles.
File:ZhangLab 2 2014-06-18 13hr 56min PC-T RRBS 15xTESTPCR.jpg  File:ZhangLab 2 2014-06-18 13hr 56min PC-T RRBS 17xTESTPCR.jpg
  • The intensity of 15X cycles is quite low compare to CTT-RRBS result (frozen tissue), which I use as the reference as the samples were derived from frozen tissue. At 17X, some samples have very faint band. Some of them look good.

2014-06-17

  • I do not want to go with too high number of cycles. I repeated experiment by doing everything almost the same as described above with minor changes.
    • This time I increase spike in unmeth lambda DNA to 1% as scRRBS experiment. The reason is I could get only 1000 reads from 10M reads of spike-in DNA 0.01%. This is too low. I expected to get 100,000 reads from 1% spike-in DNA.
    • Also, I used MethylCode kit from LifeTech as I am not sure that elution DNA 20ul (to fit volume for Zymo Lightning kit) may not sufficient to elute all DNA form AMPure beads after ligation reaction.


Msp I digestion

MspI digestion set up

Strip1 T-1_r1 T-1_r2 T-2_r1 T-2_r2 T-4_r1 T-4_r2 T-6_r1 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) 12.79 12.79 11.21 11.21 11.66 11.66 7.42 7.42
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
1ng/ul unmeth-lambda DNA 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
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 T-7_r1 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) 7.87 7.87 14.00
10X Tango Buffer 3.00 3.00 3.00
MspI 2.00 2.00 2.00
1ng/ul unmeth-lambda DNA 1.00 1.00 1.00
H2O in MspI Mix 10.00 10.00 10.00
Total (ul) 30.00 30.00 30.00

Msp I reaction Mix

Components 1x rxn 11.5 rxn Mix
MspI (10U/ul) 2.00 23.00
10x Tango Buffer 3.00 34.50
1ng/ul unmeth-lambda DNA 1 11.50
H2O 10.00 115.00
Total 16.00



End-repair/dA-tailing

  • Same as above procedures.


Methylated adaptor ligation

  • Same as above. Use the same barcode listed above


Bisulfite conversion

  • Use LifeTech MethylCode™ Bisulfite Conversion Kit
- Resuspend CT Conversion reagent with 800ul H2O, 50ul Resuspension Buffer, and 300ul DilutionBuffer
- Add 12oul of complete CT Conversion reagent to 30ul of adaptor-ligated DNA
- Elute 38ul with Elution buffer as described above.


Amplification

TEST PCR

Components Conc unit Final conc./amount unit Volume (ul) 13 rxn mix
Bis-cvt DNA 6.00 0.00
10X Reaction buffer 10 X 1 X 2.00 26.00
dNTP mix 10 mM 0.25 mM 0.50 6.50
TruS_F/R 10 uM 0.3 uM 0.60 7.80
50X SYBG 50 X 0.8 X 0.32 4.16
PfuTurbo Cx 2.5 Unit/ul 1 unit 0.40 5.20
H2O 10.18 132.34
Total 20.00
- Aliquot 14 + 6ul DNA template
- Mix well. Split 2X of 9ul
95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X [15X & 17X] --> 72C for 2min
- Load 5ul PCR product in 12% gel and run at 250 volts for 40min
File:ZhangLab 2 2014-06-20 15hr 53min PC-T RRBS 15X TEST.jpg  File:ZhangLab 2 2014-06-20 21hr 11min PC-T RRBS 17x TEST.jpg

- I reloaded 17X cycles gel. The intensity of 15X and 17X cycles were not much different because I had ~4ul left not exactly 5ul as labeled in the gel image.
- The signal in NTC should come from increasing amount of spike-in lambda DNA to 1ng.
  • 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)
  • Do not include NTC if NTC is clean in TEST PCR
Components Conc unit Final conc./amount unit Volume (ul) 10.5 rxn mix
Bis-cvt DNA 30.00 0.00
10X Reaction buffer 10 X 1 X 10.00 105.00
dNTP mix 10 mM 0.25 mM 2.50 26.25
TruS_F/R 10 uM 0.3 uM 3.00 31.50
50X SYBG 50 X 0.8 X 1.60 16.80
PfuTurbo Cx 2.5 Unit/ul 1 unit 2.00 21.00
H2O 50.90 534.45
Total 100.00
- 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 15 cycles --> 72C for 2min
- Stopped at 15X cycles based on TEST PCR
- I pooled the two PCR replicatea (total ~90ul for each samples) and purified with 1x volume AMPure beads and eluted with 50ul EB buffer
- I did PAGE quantification by loading ul of bead-purified sequencing libraries
File:ZhangLab 2 2014-06-20 21hr 08min PC-T 100ngRRBS PQ 3ul e.jpg

PAGE quantification results

Sample Index Conc. (ng/ul) Amount in 47ul (ng) Volume for 200ng (ul)
PC-T-1_1 1 6.37 299.43 31.39
PC-T-1_2 2 4.55 213.68 43.99
PC-T-2_1 4 15.85 744.80 12.62
PC-T-2_2 5 7.27 341.89 27.49
PC-T-4_1 6 6.77 318.11 29.55
PC-T-4_2 7 6.18 290.67 32.34
PC-T-6_1 8 13.18 619.39 15.18
PC-T-6_2 12 10.99 516.72 18.19
PC-C-7_1 14 5.49 258.14 36.41
PC-C-7_2 15 5.70 267.95 35.08
  • The concentration is quite over-estimated. However, all samples were quantified in the same way and on the same gel. It should not affect the molar ratio of sequencing libraries in the pool.
- I pooled 200ng each library based on PAGE quantification and did size selection in 6 well of 5-well 6% TBE gel
File:ZhangLab 2 2014-06-22 11hr 20min PC-T rrbs-100ng PAGE-SS-1.jpg File:ZhangLab 2 2014-06-22 11hr 20min PC-T rrbs-100ng PAGE-SS-2.jpg
- I precipitated in 6tubes at -80C O/N and resuspended each tube with 10ul H2O
- I took 3ul for PAGE verification and 2ul for Qubit ssDNA HS quantification.

Sequencing library ID: NP-RRBS-PCT-100ng, Jun16, 2014
- Length: 160-360bp
- Conc. by Qubit: XX ng/ul or XX nM

Index for 100ng tumor tissue RRBS sequencing libraries

Project ID Sample type Zhang lab ID Index
UCSD-004-04 Solid tumor tissue PC-T-1_1 1
Solid tumor tissue PC-T-1_2 2
UCSD-004-05 Solid tumor tissue PC-T-2_1 4
Solid tumor tissue PC-T-2_2 5
UCSD-004-07 Solid tumor tissue PC-T-4_1 6
Solid tumor tissue PC-T-4_2 7
UCSD-004-09 Solid tumor tissue PC-T-6_1 8
Solid tumor tissue PC-T-6_2 12
UCSD-004-10 Solid tumor tissue PC-C-7_1 14
Solid tumor tissue PC-C-7_2 15