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)
   CpG Methyltranferase (M.SssI, NEB), $65
   CpG Methyltranferase (M.SssI, NEB), $65
  QIAEX II gel extraction kit (150), $172
  - These columns are cheaper than QiaQuick and the MinElute columns, but seems to have better flexibility in purifying larger fragments
  - Since I do not want to perform Covaris shearing on the DNA for BSPP, I should use the QIAEX II columns instead of the QiaQuick/MinElute columns.


== IMPORTANT note for Tet1 ==
== IMPORTANT note for Tet1 ==
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* Same for Tet1 oxidation reagent 1 and 2.
* 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.
* 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 ==
== Preparation of genomic DNA for TAB-seq ==
* Set up an in vitro methylation reaction:
* For each 1 ug of genomic DNA, add 10 ng of control DNA
  Reagent                        Volume
=== Glucosylation ===
  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:
* Protect 5hmC with beta-GT as follows:
   Reagent                        Volume
   Reagent                        Volume for one reaction
   Sheared DNA, 1-3 ug            variable
   Sheared DNA, 1-3 ug            variable
   UDP-glucose, 50X              1 uL
   UDP-glucose, 50X              1 uL
Line 54: Line 41:
   Total volume                  50 uL
   Total volume                  50 uL
* Mix well and incubate at 37C for 12 to 18 hours.
* Mix well and incubate at 37C for 12 to 18 hours.
* Clean up using 1.8X AMPure beads.
* Clean up using AmPure XP beads.
=== Oxidation: NgTet1 (NEB)===
* Dilute Fe solution 1/250 with nuclease free water to a working 2 mM solution
** Make 125 uL: 0.5 uL x 500 mM FeSO4 + 124.5 uL nuclease free water
* Prepare the NgTet1 oxidation reaction:
  T4-BGT treated DNA (500 ng)      15.5 uL
  10x NgTet1 buffer                2.0 uL
  2 mM Fe Solution                  0.8 uL
  48.3 uM NgTet1 (lot6)            1.7 uL
* Mix well and allow th reaction to proceed at 34C for 30 minutes.
* Add 1 uL proteinase K (20 mg/mL) to each reaction
* 37C for 30 minutes then 95C for 10 minutes.
* Increase volume to 75 uL and purify with Micro-Bio-spin 30 columns
* Purify with AmPure Beads. Elute in 30 uL EB.  
=== Oxidation: mTet1 (WiseGene) ===
* Prepare the mTet1 oxidation reaction:
* Prepare the mTet1 oxidation reaction:
   Reagent                        Volume
   T4-BGT treated DNA (500ng)       13.25 uL
  500 ng glucosylated DNA        variable, up to 27 uL
   Tet oxidation reagent 2           7.50 uL
  Nuclease-free water            to 27 uL
   Tet oxidation reagent 1           1.75 uL
   Tet oxidation reagent 2       15 uL
   Tet1 protein                       2.50 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
* NOTE: 500 ng DNA can yield 200-300 ng after purification
* Mix well and allow the reaction to proceed at 37C for 80 minutes.
* 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.
* 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
* Clean up using BioRad Micro Bio-Spin P30 columns.
* Purify with AmPureXP beads. Elute in 30 uL EB buffer
* The oxidized DNA can be stored at -20C for several weeks.
* The oxidized DNA can be stored at -20C for several weeks.
===Oxidation: mTet2 (NEB)===
* Dilute Fe solution 1/250 with nuclease free water to a working 2 mM solution
** Make 125 uL: 0.5 uL x 500 mM FeSO4 + 124.5 uL nuclease free water
* Prepare the mTet2 oxidation reaction:
  T4-BGT treated DNA (500 ng)      13.5 uL
  5x mTet2 buffer                  5.0 uL
  2 mM Fe Solution                  2.5 uL
  63.6 uM mTet2 (lot16)            4.0 uL
* Mix well and allow the reaction to proceed at 37C for 60 minutes.
* Add 1 uL proteinase K (20 mg/mL) to each reaction
* 37C for 30 minutes then 95C for 10 minutes.
* Increase volume to 75 uL and purify with Micro-Bio-spin 30 columns
* Purify with AmPureXP Beads. Elute in 30 uL EB.
==Bisulfite conversion==
==Bisulfite conversion==
* Perform bisulfite conversion with the EpiTect bisulfite kit.
* Perform bisulfite conversion with the EpiTect bisulfite kit.
==Verification of 5mC conversion and 5hmC protection==
* Use the low input protocol (15 uL of DNA protect buffer)
*

Latest revision as of 20:46, 17 September 2015

TAB-Seq protocol[edit]

  • 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
 QIAEX II gel extraction kit (150), $172
  - These columns are cheaper than QiaQuick and the MinElute columns, but seems to have better flexibility in purifying larger fragments
  - Since I do not want to perform Covaris shearing on the DNA for BSPP, I should use the QIAEX II columns instead of the QiaQuick/MinElute columns.

IMPORTANT note for Tet1[edit]

  • 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.

Preparation of genomic DNA for TAB-seq[edit]

  • For each 1 ug of genomic DNA, add 10 ng of control DNA

Glucosylation[edit]

  • Protect 5hmC with beta-GT as follows:
 Reagent                        Volume for one reaction
 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 AmPure XP beads.

Oxidation: NgTet1 (NEB)[edit]

  • Dilute Fe solution 1/250 with nuclease free water to a working 2 mM solution
    • Make 125 uL: 0.5 uL x 500 mM FeSO4 + 124.5 uL nuclease free water
  • Prepare the NgTet1 oxidation reaction:
 T4-BGT treated DNA (500 ng)      15.5 uL
 10x NgTet1 buffer                 2.0 uL
 2 mM Fe Solution                  0.8 uL
 48.3 uM NgTet1 (lot6)             1.7 uL
  • Mix well and allow th reaction to proceed at 34C for 30 minutes.
  • Add 1 uL proteinase K (20 mg/mL) to each reaction
  • 37C for 30 minutes then 95C for 10 minutes.
  • Increase volume to 75 uL and purify with Micro-Bio-spin 30 columns
  • Purify with AmPure Beads. Elute in 30 uL EB.

Oxidation: mTet1 (WiseGene)[edit]

  • Prepare the mTet1 oxidation reaction:
 T4-BGT treated DNA (500ng)        13.25 uL
 Tet oxidation reagent 2            7.50 uL
 Tet oxidation reagent 1            1.75 uL
 Tet1 protein                       2.50 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.
  • Purify with AmPureXP beads. Elute in 30 uL EB buffer
  • The oxidized DNA can be stored at -20C for several weeks.

Oxidation: mTet2 (NEB)[edit]

  • Dilute Fe solution 1/250 with nuclease free water to a working 2 mM solution
    • Make 125 uL: 0.5 uL x 500 mM FeSO4 + 124.5 uL nuclease free water
  • Prepare the mTet2 oxidation reaction:
 T4-BGT treated DNA (500 ng)      13.5 uL
 5x mTet2 buffer                   5.0 uL
 2 mM Fe Solution                  2.5 uL
 63.6 uM mTet2 (lot16)             4.0 uL
  • Mix well and allow the reaction to proceed at 37C for 60 minutes.
  • Add 1 uL proteinase K (20 mg/mL) to each reaction
  • 37C for 30 minutes then 95C for 10 minutes.
  • Increase volume to 75 uL and purify with Micro-Bio-spin 30 columns
  • Purify with AmPureXP Beads. Elute in 30 uL EB.

Bisulfite conversion[edit]

  • Perform bisulfite conversion with the EpiTect bisulfite kit.
  • Use the low input protocol (15 uL of DNA protect buffer)