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Dinh:5hmC Lambda Controls Protocol
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===Prepare sequencing libraries=== * Pool all the leftover PCR reactions together and add water to make total volume = 30 uL, then add 60 uL AmPure beads to purify amplicons. Allow 15 minutes for binding. * After washing and drying, leave the beads in the mixture. Add dA-tailing mixture: * dA-tailing mixture: Reagent Volume per reaction Water 27.7 uL 10X PCR buffer 3.5 uL MgCl2 (25 mM) 2.1 uL dATP (10mM) 0.7 uL JumpStart Taq Pol 1.0 uL * Incubate 70C for 40 minutes. * Purify reactions with 35 uL AmPure beads. After washing and drying, leave the beads in the mixture. Add PNK mixture. * PNK mixture: Reagent Volume per reaction Water 21.5 uL T4 PNK 1.0 uL 10X T4 ligase buffer 2.5 uL * 37C for 40 minutes. Add adaptor ligation mixture. * Adaptor ligation mixture: Reagent Volume per reaction T4 DNA ligase 1.0 uL 10X T4 ligase buffer 1.0 uL TruSeq adaptors 1.0 uL Water 7.0 uL * 16C Overnight, 65C for 20 minutes * Purify each reaction with 35 uL AmPure beads. Elute with 20 uL water. * Perform PCR on 10 uL of the purified adapted amplicons: Reagent Volume per reaction KAPA Sybr Fast 25 uL TruSeq F/R primers 1.5 uL Adapted amplicons 10 uL Water 13.5 uL * Program: 95C 3 min, (95C 10s -> 60C 30s)x10, 72C 1 min * Pool all of the amplicons together and purify with PCR cleanup columns. * Run a TBE size selection on the pooled amplicons. * Sequence each library to get 30,000 reads for each index.
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