Noi/NOTES/2014-6-29

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Preparation of RRBS sequencing libraries of plasma DNA from colon and lung cancer patients[edit]

Calendar

Sample info[edit]

Project ID Zhang lab ID Conc. (ng/ul)
UCSD-006.1 6-P_1 0.200
UCSD-006.2 6-P_2 1.100
UCSD-006.3 6-P_3 1.570
UCSD-006.4 6-P_4 0.889
UCSD-006.5 6-P_5 0.487
UCSD-006.6 6-P_6 0.249
UCSD-006.7 6-P_7 0.247
UCSD-006.8 6-P_8 0.226
UCSD-006.9 6-P_9 0.408
UCSD-006.10 6-P_10 0.333
Project ID Zhang lab ID Conc. (ng/ul)
UCSD-007.1 7-P_1 0.692
UCSD-007.2 7-P_2 1.140
UCSD-007.3 7-P_3 0.208
UCSD-007.4 7-P_4 0.202
UCSD-007.5 7-P_5 1.110
UCSD-007.6 7-P_6 0.233
UCSD-007.7 7-P_7 0.229
UCSD-007.8 7-P_8 0.167
UCSD-007.9 7-P_9 0.231
UCSD-007.10 7-P_10 0.203
  • X-Y_Z:
    • X = project ID number. 4 = pancreas, 6 = colon, 7 = lung
    • Y = sample type. T = tissue, P = plasma, S = serum
    • Z = sample ID

1) MspI digestion[edit]

  • Note that the protocol of low-input DNA (1-5ng) will be different from the STD RRBS protocol (100ng).
  • Conditions: Digest 1ng of DNA with 10U of MspI in 18ul reaction at 37C O/N (normally 3h) and heat inactivate at 65C 20min
  • For 1ng DNA input, I used 1ul (10U) of MspI.
  • I spike in 1% of unmeth lambda DNA (10pg)
  • I actually start MspI at late night of 2014-06-29, so I incubated it O/N as it doesn't hurt to incubate DNA with MspI longer than 3h.


Msp I Reaction Mix[edit]

Components 1x rxn 26 rxn Mix
MspI (10U/ul) 1.00 26.00
10x Tango Buffer 2.00 52.00
12pg/ul unmeth-lambda DNA 0.83 21.67
H2O 8.17 212.33
Total 12.00

Prep
- Aliquot 39ul of MspI reaction mix to 8-tube strip.

- Add H2O to each tube
- Add 1ng of DNA to each tube
- Add 12ul 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, O/N (~12.5h) --> 65C, 20min --> hold at 4C (set heat lid)


2) End-repair/dA-tailing[edit]

Prep
- Mix 29ul of Klenow exo- with 29ul of dA:dC:dG (stock 20mM:2mM:2mM) just right before adding to MspI digested DNA
- Aliquot 7ul of Klenow, exo- and dA:dC:dG mix to 8-tube strip.

- Add 2ul of Klenow, exo- and dA:dC:dG mix to each tube of MspI digested DNA with multichannel pipette. 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)


3) Methylated adaptor ligation[edit]

  • Conditions: Add 1ul of 1:20 diluted TruSeq adaptors (do not know actual concentration of adaptors) in 25ul ligation reaction with 6000U of T4 DNA ligase

Ligation reaction set up[edit]

Prep
- Thaw diluted TruSeq adaptor on ice (used the diluted adaptors prepared on 2014-06-20, keep it on ice all the time)
- Prepare ligation reaction mix

Ligation reaction Mix[edit]

Components 1x rxn 26x rxn
dA-tailed DNA 20.00 0.00
10X Tango buffer 0.50 13.00
HC T4 DNA ligase (30units/ul) 1.00 26.00
10mM ATP 1.25 32.50
H2O 1.25 32.50
Total 24.00

Prep
- Aliquot 13ul of ligation reaction mix to 8-tube strip.

- Add 1ul of 1:20 diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction
Sample TruSeq Index Sample TruSeq Index
6-P_1 13 7-P_1 1
6-P_2 14 7-P_2 2
6-P_3 15 7-P_3 4
6-P_4 16 7-P_4 5
6-P_5 27 7-P_5 6
6-P_6 18 7-P_6 7
6-P_7 19 7-P_7 8
6-P_8 20 7-P_8 10
6-P_9 21 7-P_9 11
6-P_10 22 7-P_10 12
6-P_NTC1 22 7-P_NTC1 12
6-P_NTC2 22 7-P_NTC2 12
- Add 4ul of ligation reaction mix with multichannel pipette
- Spin down the tube
- Mix by gentle pulse-vortexing on PCR rack 10x
- Spin down the tube
- 16C, 22h (no heat lid. Ligation time is flexible. I usually do at least 20h at 16C)
- Mix by gentle pulse-vortexing on PCR rack 5x
- Quickly spin down
- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)


Bisulfite conversion[edit]

  • Performed bisulfite conversion using LifeTech MethylCode™ Bisulfite Conversion Kit and did the same procedure following manufacturer's instruction and elute with 38ul Elution buffer.

Prep

- Prepare 3 tubes of complete CT Conversion Reagent by adding 850ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer. If CT Conversion reagent prepare with 800ul H2O, need to add 5ul of H2O to 25ul adaptor-ligated DNA and added 120ul of CT conversion reagent.
- Mix well and spin down the tube
- Put on thermomixer at 37C for 15min, 1,000rpm
- Solution should be clear
- Spin down the tube
- Add 125ul 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 transferring to new tube
- Add 30ul 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[edit]

1st round PCR (fix 12 cycles)[edit]

Components Conc unit Final conc./amount unit Volume (ul) 25 rxn mix
Bis-cvt DNA 30.00 0.00
10X Reaction buffer 10 X 1 X 5.00 125.00
dNTP mix 10 mM 0.25 mM 1.25 31.25
TruS_F/R 10 uM 0.3 uM 1.50 37.50
50X SYBG 50 X 0.8 X 0.80 20.00
PfuTurbo Cx 2.5 Unit/ul 1 unit 1.00 25.00
H2O 10.45 261.25
Total 50.00
- Aliquot 20 + 30ul BIS-DNA template
- Mix well
95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 12 cycles --> 72C for 2min
- Purify with 1X AMPure beads (incubate DNA with beads 15min, the rest steps are the same as previous experiment)
- Elute with 40ul EB Buffer
- Continue to 2nd round PCR. No PAGE verification.

2nd round PCR[edit]

OPTIMIZE PCR cycle number

  • I only TEST 9 cycles of PCR
Components Conc unit Final conc. unit Volume (ul) 26 rxn mix
Purified 1st round DNA 3.00 0.00
5X Phusion HF buffer 5 X 1 X 2.00 52.00
dNTP mix 10 mM 0.25 mM 0.25 6.50
TruS_F/R 10 uM 0.3 uM 0.30 7.80
50X SYBR 50 X 0.4 X 0.08 2.08
Phusion HF 2 unit/ul 0.10 2.60
H2O 4.27 111.02
Total 10.00
- Aliquot 7 + 3ul DNA template
- Mix well.
98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 9X cycles --> 72C for 2min
- 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
File:ZhangLab 2 2014-07-02 14hr 25min 1ngRRBS 6P 1-10 TEST-9x.jpg

File:ZhangLab 2 2014-07-02 14hr 25min 1ngRRBS 7P 1-10 TEST-9x.jpg
  • A smear for sample 6P_1-10 did not look very good for me since it 's supposed to be very sharp compare to previous experiment. I think it might be over-amplified. That's why there are s smear above the expected ranged.
  • I think 8 cycles should reduce background and increase the intensity of a smear between 150-350bp. However, I'm afraid to amplified in all of template DNA at once. I do amplification 1Xrxn of 50ul instead of 2X of 50ul as usual.
Components Conc unit Final conc. unit Volume (ul) 20.5 rxn mix
Purified 1st round DNA 15.00 0.00
5X Phusion HF buffer 5 X 1 X 10.00 205.00
dNTP mix 10 mM 0.25 mM 1.25 25.63
TruS_F/R 10 uM 0.3 uM 1.50 30.75
50X SYBR 50 X 0.4 X 0.40 8.20
Phusion HF 2 unit/ul 0.50 10.25
H2O 21.35 437.68
Total 50.00
- Aliquot 35 + 15ul DNA template
- Mix well.
98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 8X cycles --> 72C for 2min
- I load 5ul of amplified DNA at 8 cycles
File:ZhangLab 2 2014-07-02 21hr 55min 6P 1-10 8x 50ulrxn.jpg

File:ZhangLab 2 2014-07-02 21hr 55min 7P 1-10 8x 50ulrxn.jpg
  • The result looked better than 9 cycles since I can see less background and the border of a smear for samples 6P_1-10 look sharper.Then I continue to do another 50ul PCR using the same condition above.

NOTE[edit]

  • Optimization of PCR cycles in small volume (10ul) is still VERY IMPORTANT for RRBS (2-3 cycles different, like 8X cycles and 10X cycles in 2nd round PCR for 1-5ng input) to get a good result even we know roughly cycles # for different input amount.
- I pool ~80ul PCR product from 1st and 2nd times and elute with 55ul EB buffer
- I did PAGE quantification by loading 3ul of bead-purified sequencing libraries
6-P_1-10: Plasma sample from colon cancer patients
File:ZhangLab 2 2014-07-03 17hr 22min PQ 6Prrbs 1-10 3ul.jpg

7-P_1-10: Plasma sample from lung cancer patients
File:ZhangLab 2 2014-07-03 17hr 22min PQ 7Prrbs 1-10 3ul.jpg

PAGE quantification results[edit]

006-P samples

Sample Index Conc. (ng/ul) Amount in 50ul (ng)
6-P-1 13 5.18 258.95
6-P-2 14 4.84 242.15
6-P-3 15 3.18 159.17
6-P-4 16 3.82 191.13
6-P-5 27 2.07 103.50
6-P-6 18 1.85 92.26
6-P-7 19 3.04 151.94
6-P-8 20 2.88 143.84
6-P-9 21 4.71 235.50
6-P-10 22 2.59 129.26

007-P samples

Sample Index Conc. (ng/ul) Amount in 50ul (ng)
7-P-1 1 7.11 355.52
7-P-2 2 5.49 274.61
7-P-3 4 3.93 196.40
7-P-4 5 8.14 407.01
7-P-5 6 3.26 163.24
7-P-6 7 5.83 291.54
7-P-7 8 8.10 405.02
7-P-8 10 9.79 489.42
7-P-9 11 3.71 185.74
7-P-10 12 6.48 324.06


Library pooling[edit]

006-P samples[edit]

NP-RRBS-6P_1ng-Jun29

Sample Index Conc. (ng/ul) Volume for 75ng (ul)
6-P-1 13 5.18 14.48
6-P-2 14 4.84 15.49
6-P-3 15 3.18 23.56
6-P-4 16 3.82 19.62
6-P-5 27 2.07 36.23
6-P-6 18 1.85 40.65
6-P-7 19 3.04 24.68
6-P-8 20 2.88 26.07
6-P-9 21 4.71 15.92
6-P-10 22 2.59 29.01
- Add 50ul of 6X loading dye and load 73.93 in 4 well of 5-well 6% TBE gel
007-P samples[edit]

NP-RRBS-7P_1ng-Jun29

Sample Index Conc. (ng/ul) Volume for 120ng (ul)
7-P-1 1 7.11 16.88
7-P-2 2 5.49 21.85
7-P-3 4 3.93 30.55
7-P-4 5 8.14 14.74
7-P-5 6 3.26 36.76
7-P-6 7 5.83 20.58
7-P-7 8 8.10 14.81
7-P-8 10 9.79 12.26
7-P-9 11 3.71 32.30
7-P-10 12 6.48 18.52
- Add 50ul of 6X loading dye and load 67.31 in 4 well of 5-well 6% TBE gel

PAGE size selection[edit]

  • I forgot to take the picture of PAGE size-selection
- NP-RRBS-6P_1ng-Jun29: Resuspend with 12ul H2O x 4tubes --> volume 48ul, 4ul for PAGE verification
- NP-RRBS-7P_1ng-Jun29: Resuspend with 12ul H2O x 4tubes --> volume 48ul, 4ul for PAGE verification

PAGE verification[edit]

2014-07-08[edit]

Preparing sequencing library pool for IGM[edit]

  • We will do HiSeq Rapid run, PE 100+7bp
  • Spike in with 5% PhiX in each lane
1). NP_RRBS_PCP-6P_1ng_20140709 (Loaded in lane 1)
Seq. Library # of ind. Library # of read for each (M) Total # of reads (M) Loading % Conc. (nM) Ratio of % to 6P_1ng # of mole to pool (nmole) Volume to pool (ul)
NP-RRBS-6P_1ng-Jun29 10 10 100 66.67 13.79 1 120 8.70
NP-RRBS-PC_P_1ng_p1-Jun20 8 5 40 26.67 28.25 0.4 48 1.70
NP-RRBS-PC_P_1ng_p2-Jun20 2 5 10 6.67 15.90 0.1 12 0.75
Total 180 11.16
Final conc. 16.14 nM
2). NP_RRBS_NCP-7P_1ng_20140709 (Loaded in lane 2)
Seq. Library # of ind. Library # of read for each (M) Total # of reads (M) Loading % Conc. (nM) Ratio of % to 7P_1ng # of mole to pool (nmole) Volume to pool (ul)
NP-RRBS-7P_1ng-Jun29 10 10 100 71.43 16.89 1 120 7.10
NP-RRBS-NC_P_1ng_p1-Jun20 4 5 20 14.29 18.95 0.2 24 1.27
NP-RRBS-NC_P_1ng_p2-Jun20 4 5 20 14.29 15.80 0.2 24 1.52
Total 168 9.89
Final conc. 16.99 nM