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== Methylated adaptor ligation == {| {{table}} border = 1 | align="center" style="width:180px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''11rxn Mix''' |- | dA-tailed DNA||20.00||0.00 |- | 10x Ligation Buffer||3.00||33.00 |- | T4 DNA Ligase (6000U/ul)||1.00||11.00 |- | TruSeq Adaptor (15uM)||1.50||0.00 |- | H2O||4.50||49.50 |- | Total||30.00|| |} :- Add 1.5ul of 15uM (non-diluted adaptor) TruSeq adaptor to each tube :{| {{table}} class = wikitable | align="center" style="width:100px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:100px;background:#f0f0f0;"|'''Truseq Index''' |- | HC-C1||1 |- | HC-C2||2 |- | HC-C3||3 |- | HC-C4||4 |- | HC-H1||5 |- | HC-H2||6 |- | HC-H3||7 |- | HC-H4||8 |- | HC-H5||9 |- | NTC||10 |} * Final conc. of adaptor in 30ul lgation reaction is 750nM :- Add 8.5ul of ligation reaction mix with adaptors :- Spin down the tube :- Mix by gentle pulse-vortexing on PCR rack 10x :- Spin down the tube :- 16C, 24h (no heat lid) :- Mix by gentle pulse-vortexing on PCR rack 5x :- Quickly spin down :- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on) * Before continue to bisulfite conversion, purify adaptor-ligated DNA with 2x volume AMPure beads (60ul each) and resuspend the bead with 21ul EB buffer ==== <u>AMPure bead purification</u> ==== :- Add 60ul AMPure bead. Mix by pipetting 10x :- Wait for 15min :- Transfer to sit on magnet for 5min :- Wash twice with 200ul freshly prepared 80% EtOH ::- After adding 80% EtOH (1st time), wait for 30sec before pipetting up & down 5x :- Remove all 80% EtOH :- Dry the bead for 15min :- Resuspend with 21 EB Buffer :- Wait for 2min. :- Transfer to sit on magnet for 2 min and transfer 20ul of adaptor-ligated DNA to new strip tubes <u>(discard the beads)</u> :- Continue to bisulfite conversion === Bisulfite conversion === * Performed bisulfite conversion using the same procedure following manufacturer's instruction [http://www.zymoresearch.com/downloads/dl/file/id/490/d5030i.pdf| '''EZ DNA Methylation-Lightning™ Kit'''] and elute with 34ul Elution buffer. :- Add 130ul complete Lightning Converson Reagent to 30ul bead-purified adaptor-ligated DNA --> total volume 150ul :- Mix by pipetting 10X with multi-channel pipette :- Spin down the tube :- Incubate following below program (Magprep, Heated Lid) ::- 98°C for 10 minutes (DNA denaturation) ::- 54°C for 1 hours (Bisulfite conversion) ::- 4°C storage for up to 20 hours or continue to desulfonation :- Add 600ul Binding Buffer to each column :- Adjust p200 to 200ul and transfer bisulfite treated DNA to the column :- Mix by pipetting 5x :- Spin down column at 14,000 rpm for 30s :- <u>Discard spnt</u> :- Add 100ul Wash Buffer (Aliquot Wash Buffer in 5mL tube) :- Spin down column at 14,000 rpm for 30s <u>(do not discard spnt)</u> :- Add 200ul Desulfonation Buffer (aliquot Desulfonation Buffer in 2mL or 5mL tube) :- Wait for 18min :- Spin down column at 14,000 rpm for 30s <u>(do not discard spnt)</u> :- Add 200ul Wash Buffer :- Spin down column at 14,000 rpm for 30s :- <u>Discard spnt</u> :- Add 200ul Wash Buffer :- Spin down column at 14,000 rpm for 3min <u>(do not discard spnt)</u> :- Transfer column to prepared 1.5mL LoBind tube & make sure that column does not touch spnt in the collecting tube before transfering to new tube :- Add 36ul of Elution Buffer (warm Elution Buffer at 60C for 10min) :- Wait for 2min :- Spin down column at 12,000 rpm for 1min :- Continue to amplification
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