Dinh:5hmC Lambda Controls Protocol

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Preparation of the sequencing and digestion spike-in controls[edit]

  • Design primers to amplify three different regions in Lambda DNA:
  • All primers can amplify DNA at 55C with good efficiency.
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

  • 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[edit]

PCR to recover controls[edit]

  • 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[edit]

  • 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[edit]

  • 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.