Noi/NOTES/2014-7-11

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Preparation of low-input RRBS sequencing libraries with MspI/TaqI double-digestion[edit]

Calendar

  • Aim: To increase the coverage of CpG in low-CpG regions as TaqI cut at sequences T^CGA site. ApeKI (G^CWGC) is another option that had been used in some group as well, but the digestion in CutSmart Buffer is not compatible with it.
  • Dr. Zhang suggested to run test experiment on tumor DNA as it has high amount and we could get information from sequencing if the protocol works well.
  • I plan to use 6-T-1 sample and also include 1 NC-P sample since I had extra tube from Nucleospin plasma DNA extraction kit. This sample could be used to confirm and compare the result of single-digestion and double-digestion of the plasma DNA.

STATUS[edit]

  • WAITING FOR REAGENTS
  • Just received all required regents at 5pm. It's quite late to get all steps done before ligation. I will do MspI digestion O/N and continue with TaqI digestion on Saturday morning. I should get the result of PCR and PAGE verification on Sunday.

Sample list/conditions[edit]

  1. MspI single digestion
    1. NC-P-27
    2. 6-T-1
    3. NTC
  2. MspI/TaqI double-digestion
    1. NC-P-27
    2. 6-T-1
    3. NTC
  • I plan to do experiment in two replicates

Note of the protocol[edit]

  • Will be added


1.1 MspI digestion[edit]

MspI digestion set up[edit]

# Sample Conc. (ng/ul) Volume for 1ng (ul) H2O to 10ul MspI Mix Total Code
1 NC-P-27_s1 0.307 3.26 6.74 8.00 18.00 NC-27_s1
2 NC-P-27_s2 0.307 3.26 6.74 8.00 18.00 NC-27_s2
3 6-T-1_s1 0.920 1.09 8.91 8.00 18.00 6T-1_s1
4 6-T-1_s2 0.920 1.09 8.91 8.00 18.00 6T-1_s2
5 NTC-s 0.000 0.00 10.00 8.00 18.00 NTC-s
6 NC-P-27_d1 0.307 3.26 6.74 8.00 18.00 NC-27_d1
7 NC-P-27_d2 0.307 3.26 6.74 8.00 18.00 NC-27_d2
8 6-T-1_d1 0.920 1.09 8.91 8.00 18.00 6T-1_d1
9 6-T-1_d2 0.920 1.09 8.91 8.00 18.00 6T-1_d2
10 NTC-d 0.000 0.00 10.00 8.00 18.00 NTC-d

- s = single digestion (MspI)
- d = double-digestion (MspI/TaqI)

MspI Reaction Mix[edit]

Components 1x rxn 11x rxn
MspI (NEB, 20U/ul) 1.00 11.00
10x CutSmart Buffer 1.80 19.80
12pg/ul unmeth lambda DNA 0.83 9.17
H2O 4.37 48.03
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 --> hold at 4C (set heat lid)
- I skipped heat inactivation of MspI at 65C for 20min because in the next TaqI digestion reaction, it will be incubated at 65C for 3h.


1.2) TaqI digestion[edit]

No TaqI reaction Mix[edit]

Components 1x rxn 6x rxn
10x CutSmart Buffer 0.50 3.00
H2O 4.50 27.00
Total 5.00

TaqI reaction Mix[edit]

Components 1x rxn 6x rxn
TaqI (NEB (20U/ul) 1.00 6.00
10x CutSmart Buffer 0.70 4.20
H2O 3.30 19.80
Total 5.00 30.00
- Add 5ul of no TaqI or TaqI 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
- 65C, 3h --> 80C, 20min --> hold at 4C (set heat lid)


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

Prep
- Mix 12ul each of Klwnow, exo- (NEB) and dA:dC:dG mix

- Add 2ul of Klwnow, exo- and dA:dC:dG mix to each tube of MspI/TaqI 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[edit]

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

Ligation Reaction Mix[edit]

Components 1x rxn 11x rxn
dA-tailed DNA 25.00 0.00
10X CutSmart Buffer 0.50 5.50
T4 DNA ligase (NEB, 2000U/ul) 1.00 11.00
10mM ATP 3.00 33.00
1:20 diluted TruSeq Adaptor 1.00 0.00
Total 30.50
- 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, 20h (no heat lid)
- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)
- Continue to bisulfite conversion
Sample TruSeq Index
NC-P-27_s1 13
NC-P-27_s2 14
6-T-1_s1 15
6-T-1_s2 16
NTC-s 16
NC-P-27_d1 27
NC-P-27_d2 18
6-T-1_d1 19
6-T-1_d2 20
NTC-d 20

Will be updated soon[edit]

Amplification[edit]

TEST at 8 and 9 cycles[edit]

8 cycles
File:ZhangLab 2 2014-07-16 19hr 54min 8x TEST-PCR double-digestion.jpg

9 cycles with the same labeling orders
File:ZhangLab 2 2014-07-16 19hr 54min 9x TEST-PCR double-digestion-nolabeled.jpg
  • The gel image looked slightly different from previous experiment because I ran in 6% TBE gel instead of 12% TBE gel to reduce the time. The pattern of libraries from plasma DNA and tissue DNA is clearly different.
  • There is a strong distinct band ~200bp which is the repetitive-rich region as mention in the reference paper. It's very strong in MspI/TaqI double-digestion libraries. Dr. Zhang

suggested to cut this band only in tissue libraries, but not plasma DNA libraries.

  • Dr. Zhang suggested to sequence 6-T-1 sample digested with MspI and MspI/TaqI for comparison. I will sequence sample Index 16 and 19.

PAGE size selection[edit]

  • I remove the band ~200bp before cutting a smear in the range ~150-350 or a little high.
File:ZhangLab 2 2014-07-17 14hr 45min dgRRBS PAGE-SS.jpg