Dinh:TAB Protocol: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Dinh
mNo edit summary
>Dinh
 
(4 intermediate revisions by the same user not shown)
Line 28: Line 28:
* 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. (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:
* 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 61: 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 QIAEX II gel extraction column (Protocol says to use 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)