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== Preparation of enzyme mix for End repair, A-tailing, Ligation, and Methylation adapters == '''Sample list'''<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Sample code on tubes''' | align="center" style="background:#f0f0f0;"|'''Well IDs''' |- | N37-Cerebellum (CRBL)||N37-1||A1 |- | N37-Colon||N37-2||B1 |- | N37-Frontal lobe (FL)||N37-3||C1 |- | N37-Heart||N37-4||D1 |- | N37-Small intestine (SI)||N37-5||E1 |- | N37-Liver||N37-6||F1 |- | N37-Lung||N37-7||G1 |- | N37-Skeletal muscle (SM)||N37-8||H1 |- | N37-Pancrease||N37-9||A2 |- | N37-Stomach||N37-10||B2 |}<br> === End repair === Sheared gDNA 200ng in 50ul {| {{table}} | align="center" style="background:#f0f0f0;"|'''Total reaction in''' | align="center" style="background:#f0f0f0;"|'''70''' | align="center" style="background:#f0f0f0;"|'''ul''' |- | DNA ||50||ul |- | End repair reaction mix ||20||ul |}<br> {| {{table}} | align="center" style="background:#f0f0f0;"|'''KAPA End repair mix''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''10x rxn mix''' |- | 10X End repai buffer||7.00||70.00 |- | KAPA End repair enzyme||3.00||30.00 |- | H2O||10.00||100.00 |- | Total||20.00||200.00 |} * Aliquot to column #1: 20ul (A & B: 40ul) - Add 20ul of End-repair reaction mix - Mix by pipetting 20 times - Shake at 1000 rpm for 5min (lid on) - Incubate at 20C for 30min - Add 120ul of diluted AMPure beads - Mix at 750rpm for 5min, 25C (lid on) - Incubate at 25C for 5 min - Put on magnet and discard spnt - Wash twice with 140ul 80% EtOH - Dry the beads === A-tailing === * Total 30ul/reaction {| {{table}} | align="center" style="background:#f0f0f0;"|'''A-tailing reaction mix''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''10x rxn mix''' |- | KAPA 10X A-tailing buffer||3.00||30.00 |- | KAPA A-tailing enzyme||2.00||20.00 |- | H2O||25.00||250.00 |- | Total||30.00||300.00 |} * Aliquot to column #2: 30ul (A & B: 60ul) - Add 30ul of A-tailing reaction mix - Mix by pipetting 20 times - Shake at 1000 rpm for 5min (lid on) - Incubate at 30C for 30min - Add 90ul of 20%PEG/2.5M NaCl - Mix at 750rpm for 5min, 25C (lid on) - Incubate at 25C for 5 min - Put on magnet and discard spnt - Wash twice with 140ul 80% EtOH - Dry the beads === Adapter ligation === {| {{table}} | align="center" style="background:#f0f0f0;"|'''Total reaction in''' | align="center" style="background:#f0f0f0;"|'''50''' | align="center" style="background:#f0f0f0;"|'''ul''' |- | Ligation reaction mix||40||ul |- | Diluted Methylation adapters||10||ul |}<br> {| {{table}} | align="center" style="background:#f0f0f0;"|'''Ligation reaction mix''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''10x rxn mix''' |- | KAPA 5X Ligation buffer||10.00||100.00 |- | KAPA DNA ligase||3.00||30.00 |- | H2O||27.00||270.00 |- | Total||40.00||400.00 |} * Aliquot ligation reaction mix to column #3: 40ul (A & B: 80ul) '''Methylation adapter preparation'''<br> 1. Use the methylated adapter with paired-end PCR primer pairs(PCR primer PE1.0 and PCR primer PE 2.0) provided in illumina's paired end genomic DNA sample prep kit 2. Use 10ul of methylated adapter to 5ug gDNA insert, if starting DNA amount is less than 5ug, titrate the volume of adapter reagent accordingly to maintain 10:1 ratio of DNA Since I used 0.2ug of DNA, used 0.2ul of adapters, but dilute to final volume 10ul (This volume is larger than KAPA protocol, but the total amount is the same) {| {{table}} | Prepare adapter||0.2ulx 10rxn||2.00 |- | H2O||9.8ulx10rxn||98.00 |- | Total||||100 |} * Aliquot diluted methylation adapter to column #4: 10ul (A & B: 20ul) - Add 40ul of Ligation reaction mix - Mix by pipetting 10 times - Add 10ul of diluted Methylation adapters - Mix by pipetting 10 times - Shake at 1000 rpm for 5min (lid on) - Incubate at 20C for 15min - Add 50ul of 20%PEG/2.5M NaCl - Mix at 750rpm for 5min, 25C (lid on) - Incubate at 25C for 5 min - Put on magnet and discard spnt - Wash twice with 140ul 80% EtOH - Dry the beads - Resuspend adapter ligated DNA with ~23ul EB buffer
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