Noi/NOTES/2014-6-20

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Preparation of low-input (1ng) RRBS sequencing libraries of cell-free DNA (plasma samples) from 10 pancreastic cancer patients and 10 normal control

Sample info

1) MspI digestion

  • Note that I had condition of MspI digestion of this experiment different from the low-input experiment (5ng) on 06-03-2014 since I used 2ul of MspI in 18ul reaction. I need to increase volume of of Klenow exo- reaction and ligation to 25 and 30ul, respectively to reduce concentration of glycerol in the reaction as there is no purification steps prior to bisulfite conversion.
  • For the future, I do not need to use 2ul of MspI as 1ul showed similar activity to 2ul based on agarose gel analysis. I can use the same condition as experiment on 06-03-2014
  • I spike in 1% of unmeth lambda DNA. This mean that I included 10pg of lambda DNA for 1ng input DNA.

MspI digestion set up

1.1) PC-P samples

# Sample Original conc. (ng/ul) Volume for 1ng (ul) H2O to 10ul MspI Mix Total Code
1 PC-P_1 0.473 2.11 7.89 8.00 18.00 P_1
2 PC-P_2 0.852 1.17 8.83 8.00 18.00 P_2
3 PC-P_3 1.360 0.74 9.26 8.00 18.00 P_3
4 PC-P_4 0.340 2.94 7.06 8.00 18.00 P_4
5 PC-P_5 0.813 1.23 8.77 8.00 18.00 P_5
6 PC-P_6 0.767 1.30 8.70 8.00 18.00 P_6
7 PC-P_7 0.644 1.55 8.45 8.00 18.00 P_7
8 PC-P_8 0.524 1.91 8.09 8.00 18.00 P_8
9 PC-P_9 0.238 4.20 5.80 8.00 18.00 P_9
10 PC-P_10 0.370 2.70 7.30 8.00 18.00 P_10
11 NTC_1 0.000 0.00 10.00 8.00 18.00 P_11
12 NTC_2 0.000 0.00 10.00 8.00 18.00 P_12

1.2) NC-P samples

# Sample Original conc. (ng/ul) Volume for 1ng (ul) H2O to 10ul MspI Mix Total Code
1 NC-1 0.293 3.41 6.59 8.00 18.00 N_1
2 NC-2 0.325 3.08 6.92 8.00 18.00 N_2
3 NC-3 0.538 1.86 8.14 8.00 18.00 N_3
4 NC-5 0.156 6.41 3.59 8.00 18.00 N_5
5 NC-6 0.148 6.76 3.24 8.00 18.00 N_6
6 NC-7 0.245 4.08 5.92 8.00 18.00 N_7
7 NC-8 0.141 7.09 2.91 8.00 18.00 N_8
8 NC-9 0.171 5.85 4.15 8.00 18.00 N_9
9 NC-10 0.134 7.46 2.54 8.00 18.00 N_10
10 NC-11 0.152 6.58 3.42 8.00 18.00 N_11
11 NTC_1 0.000 0.00 10.00 8.00 18.00 N_12
12 NTC_2 0.000 0.00 10.00 8.00 18.00 N_13

MspI Reaction Mix

Components 1x rxn 26x rxn
MspI 2.00 52.00
10x Tango Buffer 2.00 52.00
12pg/ul unmeth lambda DNA 0.83 21.67
H2O 3.17 82.33
Total 8.00
- Add H2O to each tube
- Add 1ng of DNA to each tube
- Add 8ul 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

Prep

  • I increased reaction volume to 25ul as reason above
Components 1x rxn 28x rxn
MspI-digested DNA 18.00 0.00
10X Tango Buffer 0.50 14.00
Klenow, exo- 1.00 28.00
dA:dC:dG 1.00 28.00
H2O 4.50 126.00
Total 25.00 196.00
- Aliquot 24.50 of Klwnow, exo- and dA:dC:dG reaction mix to 8-tube strip
- Add 7ul of Klwnow, exo- and dA:dC:dG reaction 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)
  • No AMPure bead purification

3) Methylated adaptor ligation

  • Add 1ul of 1:20 diluted TruSeq adaptors to each end-repaired DNA in total reaction volume 30ul

Ligation reaction set up

Prep
- Dilute 1:20 TruSeq adaptor by adding 2ul of TruSeq adaptor to 40ul H2O

Ligation reaction Mix

Components 1x rxn 26x rxn
dA-tailed DNA 25.00 0.00
10X Tango buffer 0.50 13.00
HC T4 DNA ligase (30units/ul) 1.00 26.00
10mM ATP 1.50 39.00
H2O 1.00 26.00
Total 29.00
- Aliquot 13ul of ligation reaction mix to 8-tube strip

PC-P samples

Sample Index
PC_P_1 1
PC_P_2 2
PC_P_3 4
PC_P_4 5
PC_P_5 6
PC_P_6 7
PC_P_7 8
PC_P_8 10
PC_P_9 11
PC_P_10 12
NTC_1 11
NTC_2 12

NC-P samples

Sample Index
NC-1 13
NC-2 14
NC-3 15
NC-5 16
NC-6 27
NC-7 18
NC-8 19
NC-9 20
NC-10 21
NC-11 22
NTC_1 21
NTC_2 22
- Add 1ul of 1:20 diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction
- Add 4ul of ligation reaction mix
- Spin down the tube
- Mix by gentle pulse-vortexing on PCR rack 10x
- Spin down the tube
- 16C, 30h (no heat lid) --> I had no time to come back to continue to next step. I incubate longer.
- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)
- Continue to bisulfite conversion

4) Bisulfite conversion

  • Performed bisulfite conversion using the same procedure following manufacturer's instruction and elute with 30ul Elution buffer.

Prep

- Prepare 2 tubes of complete CT Conversion Reagent by adding 800ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer
- Mix well and spin down the tube
- Put on thermomixer at 37C for 15min, 1000rpm
- Solution should be clear
- Spin down the tube
- 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 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

1st round PCR (12X cycles

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

1st round PCR (12X cycles)

OPTIMIZE PCR cycle number

  • I optimize PCR cycle number between 10-16 cycles by doing PCR in small volume (~10ul) and stopped reaction at 10X, 12X, 14X, and 16X cycles.
  • Then analyze in 12% TBE gel
  • Select the optimum cycle number to amplify the rest of 1st round PCR

PC-P smaple

Components Conc unit Final conc. unit Volume (ul) 13 rxn mix
Purified 1st round DNA 12.00 0.00
5X Phusion HF buffer 5 X 1 X 8.00 104.00
dNTP mix 10 mM 0.25 mM 1.00 13.00
TruS_F/R 10 uM 0.3 uM 1.20 15.60
50X SYBR 50 X 0.4 X 0.32 4.16
Phusion HF 2 unit/ul 0.80 10.40
H2O 16.68 216.84
Total 40.00
- Aliquot 28 + 12ul DNA template
- Mix well. Split 4X of 8ul
98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X [10X, 12X, 14X, 16X] 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