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=== Methylated adaptor ligation === - To avoid the overlapping of indexes to some scRRBS libraries that may need to sequence in the same run (HiSeq Rapid run has only 2 lanes), I will not use index 2, 4, 5, and 12<br> - I will use 1ul of 1:10 diluted TruSeq adaptors for both low-input RRBS and STD RRBS.<br> '''Index list'''<br> {| {{table}}class = wikitable | align="center" style="width:80px;background:#f0f0f0;"|'''CCT-5ng_1''' | align="center" style="width:80px;background:#f0f0f0;"|'''CTT-5ng_2''' | align="center" style="width:80px;background:#f0f0f0;"|'''CTT-F-5ng_1''' | align="center" style="width:80px;background:#f0f0f0;"|'''CTT-F-5ng_2''' | align="center" style="width:80px;background:#f0f0f0;"|'''5ng NTC''' |- | Ind_9||Ind_10||Ind_11||Ind_13||Ind_14 |} {| {{table}} class = wikitable | align="center" style="width:100px;background:#f0f0f0;"|'''CCT-100ng_1''' | align="center" style="width:100px;background:#f0f0f0;"|'''CTT-100ng_2''' | align="center" style="width:100px;background:#f0f0f0;"|'''CTT-F-100ng_1''' | align="center" style="width:100px;background:#f0f0f0;"|'''CTT-F-100ng_2''' | align="center" style="width:100px;background:#f0f0f0;"|'''100ng NTC''' |- | Ind_15||Ind_16||Ind_18||Ind_19||Ind_20 |} '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Diluted 1:10 of TrueSeq methylated adaptors by mixing 1ul of adaptor with 10ul H2O and mix - Prepare ligation reaction mix :{| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''11x rxn mix''' |- | dA-tailed reaction||20.00||0.00 |- | 10X Tango buffer||0.50||5.50 |- | HC T4 DNA ligase (30units/ul)||1.00||11.00 |- | 10mM ATP ||1.25||13.75 |- | H2O||1.25||13.75 |- | Total||24.00||44.00 |} :- Add 1ul of diluted methylated adapter :- Add 4ul of ligation reaction mix :- Spin down the tube :- Mix by gentle pulse-vortexting for 10x :- Spin down the tube :- ''Incubate at 16C for 18h (no heat lid)'' :- ''Heat inactivate at 65C for 20min'' '''2014-06-03, continued'''<br> ==== Low-input RRBS ==== :- Heat inactivate at 65C, 20min :- Wait for STD RRBS sample for AMPure bead purification ==== STD RRBS ==== - AMPure bead purification with 2X volume of the bead :- Mix 50ul of AMPure beads with 25ul ligated DNA. Mix by pipetting 10x :- Sit for 30min :- Transfer to sit on magnet for 5min :- Wash twice with 160ul freshly prepared 75% EtOH :- Dry the bead for 3-5min :- Resuspend with 27 H2O :- Transfer to new tube for bisulfite conversion. 25ul will be used for bisulfite conversion.
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