Dinh:TAB Protocol: Difference between revisions

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   Proteinase K
   Proteinase K
* Misc:
* Misc:
   Micro Bio-Spin 30 columns (BioRad) .. may use P 6 columns instead?
   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
   EpiTect Bisulfite Kit
   PfuTurbo Cx Hotstart DNA polymerase (agilent)
   PfuTurbo Cx Hotstart DNA polymerase (agilent)

Revision as of 23:56, 29 December 2014

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.

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 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