Dinh:TAB Protocol

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Revision as of 00:29, 30 December 2014 by >Dinh (→‎Preparation of genomic DNA)
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TAB-Seq protocol

  • This protocol cannot start from methylated adaptor ligated libraries. The 5mC would be converted to T.
  • Protocol guides:
 Nature protocol for Tet-assisted bisulfite sequencing of 5hmC : http://www.nature.com/nprot/journal/v7/n12/abs/nprot.2012.137.html
 Manual for kits
  • T4 Phage β-glucosyltransferase (T4-BGT) from NEB ($65.63)
 T4 Phage β-glucosyltransferase
 NEB buffer 4 (10x)
 Uridine Diphosphate Glucose (50X)
  • Tet1 Oxidation kit from Wisegene ($669 + $90 shipping)
 Tet1 oxidation reagent 1
 Tet1 oxidation reagent 2
 Tet1 protein
 Proteinase K
  • Misc:
 Micro Bio-Spin 30 columns (BioRad)
  - By gravitational force, larger fragments will get pulled down first, smaller fragments are retained in the gel.
  - Use P6 columns may be better because it have a smaller pore size, so cleaner separation can be achieved.
 EpiTect Bisulfite Kit
 PfuTurbo Cx Hotstart DNA polymerase (agilent)
 CpG Methyltranferase (M.SssI, NEB), $65

IMPORTANT note for Tet1

  • Upon first use, aliquot Tet1 into single use portions. Thaw/Freeze up to only 3 times!
  • Same for Tet1 oxidation reagent 1 and 2.
  • The reaction solution should turn from transparent to slightly brown after mixing Tet oxidation reagents 1 and 2.

Creating methylated lambda DNA for spike in control

  • Set up an in vitro methylation reaction:
 Reagent                         Volume
 Milli-Q water                   44 uL
 NEBuffer 2 (10x)                10 uL
 S-adenosylmethionine (SAM)       2 uL
 Unmethyl- lambda DNA 450ng/ul   40 uL
 SssI methylase ( 20 U/uL)        4 uL
 -------------------------------------
 Final volume                    100 uL
  • Mix well and incubate at 37C for 2 hours. Heat inactivate at 65C for 20 min.
  • NOTE: Use fresh SAM.
  • Clean up the DNA with the QIAEX II gel extraction kit and elute methylated DNA in 20 uL Milli-Q water. (NOTE: Do not use QiaQuick columns because they can only elute up to 10 kbp).

Preparation of genomic DNA

  • For each 1 ug of genomic DNA, add 5 ng of methylated lambda DNA control.
  • Fragment the DNA (recommended to use Covaris):
    • For RRBS may use MspI digestion to fragment the DNA
    • For BSPP may use needle shearing to get 25 kbp fragments
  • May add 5hmC control generated by PCR for the oxBS protocol to sheared DNA.
  • Purify sheared DNA with the QIAEX II gel extraction columns. (Protocol says to use Qiagen MinElute columns...).

Preparation of Tet-assisted oxidation

  • Protect 5hmC with beta-GT as follows:
 Reagent                        Volume
 Sheared DNA, 1-3 ug            variable
 UDP-glucose, 50X               1 uL
 NEBuffer 4, 10X                5 uL
 Nuclease-free water            to 49 uL
 T4-BGT, 10U/uL                 1 uL
 -------------------------------------
 Total volume                   50 uL
  • Mix well and incubate at 37C for 12 to 18 hours.
  • Clean up using 1.8X AMPure beads.
  • Prepare the mTet1 oxidation reaction:
 Reagent                        Volume
 500 ng glucosylated DNA        variable, up to 27 uL
 Nuclease-free water            to 27 uL
 Tet oxidation reagent 2        15 uL
 Tet oxidation reagent 1        3.5 uL
 Tet1 protein                   5.0 uL
 ---------------------------------------
 Total                          50 uL
  • NOTE: 500 ng DNA can yield 200-300 ng after purification
  • Mix well and allow the reaction to proceed at 37C for 80 minutes.
  • Add 1 uL proteinase K (20 mg/mL) to the reaction mixture and incubate at 50C for 1 hour.
  • Clean up using BioRad Micro Bio-Spin P30 columns and then a Qiagen QIAquick column. Elute in 30 uL EB buffer
  • The oxidized DNA can be stored at -20C for several weeks.

Bisulfite conversion

  • Perform bisulfite conversion with the EpiTect bisulfite kit.

Verification of 5mC conversion and 5hmC protection