Noi/NOTES/2014-7-11
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Preparation of low-input RRBS sequencing libraries with MspI/TaqI double-digestion[edit]
- 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]
- MspI single digestion
- NC-P-27
- 6-T-1
- NTC
- MspI/TaqI double-digestion
- NC-P-27
- 6-T-1
- 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.
- - 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