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= Preparation of WGBS of cfDNA from plasma using KAPA Hyper Prep kit = [[noi:DMR220k_LabNotes#2014|'''Link to calendar''']] * Rational: Will be added * [[Media:KAPA_Hyper_Prep_Kit_TDS.pdf|'''KAPA Hyper Prep Kit Manual''']] '''Sample list'''<br> '''No.''' '''Sample IDs''' '''Conc. (ng/ul)''' #1 NC-2 0.325 #2 NC-3 0.538 #3 6P-2 1.10 #4 6P-3 1.57 * Note: I will reduce the reaction volume to be half of the instruction and reduce the reagent volume/amount proportionally. * Actual reaction set up: :- 50ul of fragmented DNA :- 7ul of End repair & dA-tailing buffer :- 3ul of End repair & dA-tailing enzyme mix :- Total reaction: 60ul == End repair & dA-tailing == ==== End repair & dA-tailing reaction mix ==== {| {{table}} border = 1 | align="center" style="width:200px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume''' | align="center" style="width:80px;background:#f0f0f0;"|'''half rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''5.2x rxn''' |- | End repair/dA-tailing buffer ||7.00||3.50||18.20 |- | End repair/dA-tailing enzyme mix||3.00||1.50||7.80 |- | Total||10.00||5.00|| |} ==== Reaction set up ==== {| {{table}} border = 1 | align="center" style="width:140px;background:#f0f0f0;"|'''Sample IDs''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 1ng (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''End repair/dA-tailing rxn mix''' | align="center" style="width:80px;background:#f0f0f0;"|'''H2O (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total (ul)''' |- | NC-2||0.33||3.08||5.00||21.92||30.00 |- | NC-3||0.54||1.86||5.00||23.14||30.00 |- | 6P-2||1.10||0.91||5.00||24.09||30.00 |- | 6P-3||1.57||0.64||5.00||24.36||30.00 |- | NTC||0.00||0.00||5.00||25.00||30.00 |} :- Mix the reaction mix well :- 20C, 30min :- 65C, 30min, :- Hold 4C :- Proceed to Adaptor ligation immediately == Adaptor ligation == ==== Adaptor ligation reaction mix ==== {| {{table}} border = 1 | align="center" style="width:200px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume''' | align="center" style="width:80px;background:#f0f0f0;"|'''half rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''5.2x rxn''' |- | Ligation buffer||30.00||15.00||78.00 |- | DNA ligase||10.00||5.00||26.00 |- | H2O||8.00||4.00||20.80 |- | Total||48.00||24.00|| |} ==== Adaptor ligation reaction set up ==== {| {{table}} border = 1 | align="center" style="width:200px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' |- | End repair & dA-tailing rxn product||30.00 |- | 1:20 diluted adaptor (750nM)||1.00 |- | Adaptor ligation rxn mix||24.00 |- | Total||55.00 |} :- Add 1ul of 1:20 diluted M-adaptor :- Add 24ul of adaptor ligation reaction mix :- Mix well :- 20C, 15min :- Continue to SPRI clean up immediately * Final conc. of adaptor in 55ul reaction is ~13.8nM, which is very close to 14nM recommended by the kit. ==== <u>1X AMPure bead purification</u> ==== * Actual protocol uses 0.8X AMPure beads '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Freshly prepare 5mL of 80% EtOH by mixing 4mL of 100% EtOH with 1mL of H2O<br> - Aliquot 280uL of resuspened AMPure bead in 1.5mL tube :- Add 55ul AMPure bead. Mix by pipetting 10x :- Sit for 15min :- Transfer to sit on magnet for 5min :- Wash twice with 200ul freshly prepared 80% EtOH :- Dry the bead for 3-5min :- Resuspend with 21ul EB buffer :- Continue to bisulfite conversion immediately * Note: I paused at this step by putting adaptor-logated DNA on ice ~3h to wait for the reactiosn from NEXTflex cell free DNA Seq protocol to continue to bisulfite conversion together. <br>
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