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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> = Preparation of WGBS of cfDNA from plasma using NEXTflex Cell Free DNA Seq Kit = * The kit just arrived today, and I want to finish experiment ASAP. I then continue to do experiment using the same samples as of KAPA Hyper Prep kit * I set up the reaction similar to KAPA Hyper Prep kit by reducing total volume/amount of reagent in each step to a half of instruction of the kit == Bisulfite conversion == * I performed bisulfite conversion using the same procedure following manufacturer's instruction of '''EZ DNA Methylation-Lightning kit''' and elute with 34ul elution buffer. '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> :- Aliquot 6.12mL of binding buffer & mix with 5.2ul of 10ng/ul tRNA :- Aliquot 5.1mL of Washing buffer :- Aliquot 2.1mL of Desulfonation buffer :- Aliquot 340ul of Elution buffer == Amplification == * For amplification, I can not use KAPA HIFI HotStart Ready Mix since I do bisulfite conversion on the DNA. I will use Pfu Turbo Cx for amplification. * I normally do 2 round of PCR for the input less than 10ng. In this experiment, I will do a quick test single round PCR in small volume and run PCR for 18 & 20 cycles. The kit recommend 17-19 cycles for 1ng input of normal sheared gDNA. I will increase few more cycles for bisulfite converted DNA. '''Adapted PCR set up in 50ul''' '''Components 1x rxn''' 10X Reaction Buffer 5.00 10mM dNTP mix 1.25 10X Lib Amp Primer mix 5.00 Adaptor ligated library 30.00 Pfu Turbo Cx 1.00 H2O 7.75 Total 50.00 * For the quick test, I will use a little less of DNA template
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