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= Preparation of low-input RRBS sequencing libraries with MspI/TaqI double-digestion = [[noi:DMR220k_LabNotes|'''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 === * 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 ==== # 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 == * Will be added <br> == 1.1 MspI digestion == ==== <u>MspI digestion set up</u> ==== {| {{table}} class = wikitable | align="center" style="width:40px;background:#f0f0f0;"|'''#''' | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:70px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:70px;background:#f0f0f0;"|'''Volume for 1ng (ul)''' | align="center" style="width:70px;background:#f0f0f0;"|'''H2O to 10ul''' | align="center" style="width:70px;background:#f0f0f0;"|'''MspI Mix''' | align="center" style="width:70px;background:#f0f0f0;"|'''Total''' | align="center" style="width:80px;background:#f0f0f0;"|'''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)<br> - d = double-digestion (MspI/TaqI)<br> ==== MspI Reaction Mix ==== {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''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.''<br> <br> == 1.2) TaqI digestion == ====<u>No TaqI reaction Mix</u> ==== {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''6x rxn''' |- | 10x CutSmart Buffer||0.50||3.00 |- | H2O||4.50||27.00 |- | Total||5.00|| |} ====<u>TaqI reaction Mix</u>==== {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''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)<br> <br> == 2) End-repair/dA-tailing == '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - 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<br> == 3) Methylated adaptor ligation == * Add 1ul of 1:20 diluted TruSeq adaptors to each end-repaired DNA in total reaction volume 30ul ==== <u>Ligation Reaction Mix</u> ==== {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:70px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:70px;background:#f0f0f0;"|'''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 :{| {{table}} class = wikitable | align="center" style="width:100px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''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 === == Amplification == === TEST at 8 and 9 cycles === '''8 cycles''' [[File:ZhangLab_2 2014-07-16 19hr 54min_8x_TEST-PCR_double-digestion.jpg| 400px]] '''9 cycles''' with the same labeling orders [[File:ZhangLab_2 2014-07-16 19hr 54min_9x_TEST-PCR_double-digestion-nolabeled.jpg| 400px]] * 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 ==== * 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| 300px]]
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