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Dinh:5hmC Lambda Controls Protocol
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== Preparation of the sequencing and digestion spike-in controls== * Design primers to amplify three different regions in Lambda DNA: * All primers can amplify DNA at 55C with good efficiency. <nowiki>Lambda_B_T_F TAGGAAGATAGTGTTTATGT Lambda_B_T_R CCAACAAAAATTTCTCCTAT Lambda_B_C_F CAGGAAGACAGTGCTCATGC Lambda_B_C_R CCAGCAGGGATTTCTCCTGT Lambda_E_T_F TGTTATTTATGTTGTATGGTGT Lambda_E_T_R CAACTAACTTCTTTTCTTTCAC Lambda_E_C_F TGTTATTCATGTTGCATGGTGC Lambda_E_C_R CAGCTGACTTCTTTTCTTTCAC Lambda_F_T_F TTGTTTATAGGAGATATGGTAGA Lambda_F_T_R CTTACTAACCAATTCCTAAA Lambda_F_C_F ttgctcataggagatatggt Lambda_F_C_R cttgctaaccaattcctagg </nowiki> * Use the "C" primers to amplify lambda DNA with ZymoTaq polymerase * Perform size selection of the amplicons. IMPORTANT to prevent trace amount of unmodified DNA to remain in the mixtures. * Use size selected amplicons with different dNTP mixtures containing: dCTPs, dmCTPs, or dhmCTPs. * Reaction set up: Reagent Volume per reaction Reaction buffer 25 uL dNTPs mixture 5 uL (for 5hmC mixture (2.5mM), and 1.25 uL for C/mC mixture (10 mM) Primers (F/R, 10uM) 2 uL Template (10 nM) 1 uL Nuclease-free water to 50 uL Zymo Taq Polymerase 0.4 uL * Program: 95C (10min), (95C 30s -> 55C 30s -> 72C 1 min)x30, 72C 7 min * Purify each mixture with PCR cleanup columns and quantify with Qubit. B - 5hmC 27.5 ng/uL E - 5mC 25.6 ng/uL F - C 14.4 ng/uL == Quality control check: conversion efficiency== ===PCR to recover controls=== * Prepare PCR on the converted DNA using a small amount of input: Reagent Volume per reaction KAPA HiFi Uracil 5.0 uL Primer F/R ( 10 uM) 1.5 uL Water 3.0 uL Template 0.5 uL * IMPORTANT: primers to use - 5hmC controls - for oxBS - use both "C" primers - for BS - use F "T" and R "C" C controls - (all) use both "T" primers mC controls - (all) use F "T" and R "C" * Program: 98C 45s, (98C 15s -> 55C 30s -> 72C 30s)x30, 72C 1 min ===Digestion test=== * Use 1 uL of amplicons for SphI digestion Reagent Volume per reaction SphI 0.5 uL Template 1.0 uL NEBbuffer2.1 1.0 uL Water 7.5 uL * 3.5 hr at 37C, 65C for 20 min. * Run all of digestion reactions on a PAGE gel. ===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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