Dinh:TAB Protocol: Difference between revisions
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>Dinh |
>Dinh |
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(9 intermediate revisions by the same user not shown) | |||
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Proteinase K | Proteinase K | ||
* Misc: | * Misc: | ||
Micro Bio-Spin 30 columns (BioRad) .. | 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 == | ||
Line 23: | 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. | ||
== Preparation of genomic DNA for TAB-seq == | |||
* For each 1 ug of genomic DNA, add 10 ng of control DNA | |||
=== Glucosylation === | |||
== Preparation of genomic DNA == | |||
* For each 1 ug of genomic DNA, add | |||
== | |||
* 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. | * 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: | ||
T4-BGT treated DNA (500ng) 13.25 uL | |||
Tet oxidation reagent 2 7.50 uL | |||
Tet oxidation reagent 1 1.75 uL | |||
Tet oxidation reagent 2 | Tet1 protein 2.50 uL | ||
Tet oxidation reagent 1 | |||
Tet1 protein | |||
* 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 | * 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. | ||
* 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)