Noi/NOTES/2014-6-16: Difference between revisions
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:- I precipitated in 6tubes at -80C O/N and resuspended each tube with 10ul H2O | :- 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. | :- I took 3ul for PAGE verification and 2ul for Qubit ssDNA HS quantification. | ||
'''Sequencing library ID: NP-RRBS-PCT-100ng, Jun16, 2014''' | '''Sequencing library ID: NP-RRBS-PCT-100ng, Jun16, 2014'''<br> | ||
- Length: 160-360bp<br> | |||
- Conc. by Qubit: XX ng/ul or XX nM | |||
==== <span style="color:blue"><u>Index for 100ng tumor tissue RRBS sequencing libraries</u></span> ==== | ==== <span style="color:blue"><u>Index for 100ng tumor tissue RRBS sequencing libraries</u></span> ==== |
Revision as of 18:41, 22 June 2014
Preparation of RRBS sequencing libraries of solid tumor tissue sample from pancreatic cancer patients
Alan will perform this 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 | 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 the same as described above.
- This time I increase spike in unmeth lambda DNA to 1% as scRRBS experiment. In addition, 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 |