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=== 5) Methylated adaptor ligation === * Ligate A-tailed DNA with 1ul of 1:25 diluted Illumina indexed methylated adaptor (TruSeq adaptor) in total reaction 25ul at 16C for 30min and 4C for at least 16h. ** In Dr. Tang's paper, he used 1ul of 1:20 diluted TruSeq adaptor * We have modified to incubate at 16C O/N * For this round of experiment, Youjin preferred to use STD Illumina adaptor as he wanted to include all samples in the same lane for sequencing in case that the experiment works well. {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''36x rxn mix''' |- | dA-tailed reaction||20.00||0.00 |- | 10X Tango buffer||0.50||18.00 |- | HC T4 DNA ligase (30units/ul)||1.00||36.00 |- | 10mM ATP||1.25||45.00 |- | 15uM Univ adaptor||0.02||0.72 |- | H2O||2.23||80.28 |- | Total||25.00|| |} :- Add 5ul of ligation reaction mix with 12nM of STD Illumina adaptor :- Spin down the plate at 2000rpm for 2min :- Mix by gentle pulse-vortexting for 10x :- Spin down the plate at 2,000rpm for 3min :- ''Incubate at 16C O/N (~18h, no heat lid)'' :- ''Heat inactivate at 65C for 20min''. This time I skipped this step :- Spin down the plate at 2,000rpm for 1min before continuing to next step <br>
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