Noi/NOTES/2012-8-26: Difference between revisions
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>Noi |
>Noi m (→End repair) |
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| Total||20.00||200.00 | | Total||20.00||200.00 | ||
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* Aliquot to column #1: 20ul ( | * Aliquot to column #1: 20ul (A & B: 40ul) | ||
- Add 20ul of End-repair reaction mix | - Add 20ul of End-repair reaction mix | ||
- Mix by pipetting 20 times | - Mix by pipetting 20 times | ||
Line 64: | Line 64: | ||
- Wash twice with 140ul 80% EtOH | - Wash twice with 140ul 80% EtOH | ||
- Dry the beads | - Dry the beads | ||
=== A-tailing === | === A-tailing === | ||
* Total 30ul/reaction | * Total 30ul/reaction |
Revision as of 09:04, 28 August 2012
WGBS library preparation of low input DNA N37 10 tissues samples using KAPA protocol (from Eppendorf) and epMotion
Preparation of enzyme mix for End repair, A-tailing, Ligation, and Methylation adapters
Sample list
Sample IDs | Sample code on tubes | Well IDs |
N37-Cerebellum (CRBL) | N37-1 | A1 |
N37-Colon | N37-2 | B1 |
N37-Frontal lobe (FL) | N37-3 | C1 |
N37-Heart | N37-4 | D1 |
N37-Small intestine (SI) | N37-5 | E1 |
N37-Liver | N37-6 | F1 |
N37-Lung | N37-7 | G1 |
N37-Skeletal muscle (SM) | N37-8 | H1 |
N37-Pancrease | N37-9 | A2 |
N37-Stomach | N37-10 | B2 |
End repair
Sheared gDNA 200ng in 50ul
Total reaction in | 70 | ul |
DNA | 50 | ul |
End repair reaction mix | 20 | ul |
KAPA End repair mix | 1x rxn | 10x rxn mix |
10X End repai buffer | 7.00 | 70.00 |
KAPA End repair enzyme | 3.00 | 30.00 |
H2O | 10.00 | 100.00 |
Total | 20.00 | 200.00 |
- Aliquot to column #1: 20ul (A & B: 40ul)
- Add 20ul of End-repair reaction mix - Mix by pipetting 20 times - Shake at 1000 rpm for 5min (lid on) - Incubate at 20C for 30min - Add 120ul of diluted AMPure beads - Mix at 750rpm for 5min, 25C (lid on) - Incubate at 25C for 5 min - Put on magnet and discard spnt - Wash twice with 140ul 80% EtOH - Dry the beads
A-tailing
- Total 30ul/reaction
A-tailing reaction mix | 1x rxn | 10x rxn mix |
KAPA 10X A-tailing buffer | 3.00 | 30.00 |
KAPA A-tailing enzyme | 2.00 | 20.00 |
H2O | 25.00 | 250.00 |
Total | 30.00 | 300.00 |
- Aliquot to column #2: 30ul (1 & 2: 60ul)
- Add 30ul of A-tailing reaction mix - Mix by pipetting 20 times - Shake at 1000 rpm for 5min (lid on) - Incubate at 30C for 30min - Add 90ul of 20%PEG/2.5M NaCl - Mix at 750rpm for 5min, 25C (lid on) - Incubate at 25C for 5 min - Put on magnet and discard spnt - Wash twice with 140ul 80% EtOH - Dry the beads
Adapter ligation
Total reaction in | 50 | ul |
Ligation reaction mix | 40 | ul |
Diluted Methylation adapters | 10 | ul |
Ligation reaction mix | 1x rxn | 10x rxn mix |
KAPA 5X Ligation buffer | 10.00 | 100.00 |
KAPA DNA ligase | 3.00 | 30.00 |
H2O | 27.00 | 270.00 |
Total | 40.00 | 400.00 |
- Aliquot ligation reaction mix to column #3: 40ul (1 & 2: 80ul)
Methylation adapter preparation
Prepare adapter | 0.2ulx 10rxn | 2.00 |
H2O | 9.8ulx10rxn | 98.00 |
Total | 100 |
- Aliquot diluted methylation adapter to column #4: 10ul (1 & 2: 20ul)
- Add 40ul of Ligation reaction mix - Mix by pipetting 10 times - Add 10ul of diluted Methylation adapters - Mix by pipetting 10 times - Shake at 1000 rpm for 5min (lid on) - Incubate at 20C for 15min - Add 50ul of 20%PEG/2.5M NaCl - Mix at 750rpm for 5min, 25C (lid on) - Incubate at 25C for 5 min - Put on magnet and discard spnt - Wash twice with 140ul 80% EtOH - Dry the beads - Resuspend adapter ligated DNA with ~23ul EB buffer
qPCR to check if the end repair, A-tailing, and adapter ligation work
Components | 1rxn | 11.5 rxn mix |
Adapter ligated DNA | 1.00 | 0.00 |
10uM PCR_F | 0.50 | 5.75 |
10uM PCR_R.Ind2 | 0.50 | 5.75 |
2x iQ MM | 12.50 | 143.75 |
H2O | 10.50 | 120.75 |
Total | 25.00 | 276.00 |
- Aliquot 24ul, add 1ul of adapter ligated DNA
Program 98C 2min --> (98C 15sec -> 64C 20sec -> 72C 30sec) x 12 cycles --> 72C 3min --> 15C Hold File:20120826 testqPCR adapterligated N37.png - Load 2ul of PCR product to check amplification File:20120826 testqPCR adapterligated N37.jpg
Bisulfite conversion using EZ-96 DNA Methylation-Lightning™ MagPrep
- Performed bisulfite conversion manually
- Elute with 40ul Elution buffer (final volume ~35ul)
Amplification of bisulfite sequencing libraries with N2 barcoded primers
- Perform in 50ul reaction with PCR_R.Ind1-10, add 7ul of adapter-ligated bis-cvt DNA
Components | 1rxn | 11.5 rxn mix |
Adapter ligated bis-cvt DNA | 7.00 | 0.00 |
10uM PCR_F | 1.00 | 11.50 |
10uM PCR_R.Ind1-8, 21,22 | 1.00 | 0.00 |
2x iQ MM | 25.00 | 287.50 |
H2O | 16.00 | 184.00 |
Total | 50.00 | 483.00 |
- Aliquot 42ul, add 1ul PCR_R.IndX and 7ul of adapter ligated bis-cvt DNA
Program 98C 2min --> (98C 15sec -> 64C 20sec -> 72C 45sec) x 10 cycles --> 72C 3min --> 15C Hold
qPCR result File:20120827 qPCR WGBS N37 50ul.png
- Performed qPCR in total volume 100ul by using the same condition as above (in duplicated)
Components | 1rxn | 21.5 rxn mix |
Adapter ligated bis-cvt DNA | 14.00 | 0.00 |
10uM PCR_F | 2.00 | 43.00 |
10uM PCR_R.Ind1-10 | 2.00 | 0.00 |
2x iQ MM | 50.00 | 1075.00 |
H2O | 32.00 | 688.00 |
Total | 100.00 | 1806.00 |
- Aliquot 84ul, add 2ul PCR_R.IndX and 14ul of adapter ligated bis-cvt DNA
Program 98C 2min --> (98C 15sec -> 64C 20sec -> 72C 45sec) x 10 cycles --> 72C 3min --> 15C Hold
qPCR results File:20120827 qPCR WGBS N37 rep1-2.png
Sample IDs | Labeling | Indx |
N37-Cerebellum (CRBL) | N37-1 | PCR_R.N2Indx1 |
N37-Colon | N37-2 | PCR_R.N2Indx2 |
N37-Frontal lobe (FL) | N37-3 | PCR_R.N2Indx3 |
N37-Heart | N37-4 | PCR_R.N2Indx4 |
N37-Small intestine (SI) | N37-5 | PCR_R.N2Indx5 |
N37-Liver | N37-6 | PCR_R.N2Indx6 |
N37-Lung | N37-7 | PCR_R.N2Indx7 |
N37-Skeletal muscle (SM) | N37-8 | PCR_R.N2Indx8 |
N37-Pancreas | N37-9 | PCR_R.N2Indx21 |
N37-Stomach | N37-10 | PCR_R.N2Indx22 |
- Purify all PCR products (total volume 250ul) with 0.8 volume of 3x diluted AMPure bead by EPmotion - Elute with 70ul EB buffer - Perform PAGE quantification by loading 1ul of AMPure bead purified DNA File:ZhangLab 2 2012-08-27 17hr 22min PQ bead WGBS N37.jpg
PAGE quantification of 400-600bp fragments
File:ZhangLab 2 2012-08-27 17hr 40min PQ 400-600bp.jpg
Sample IDs | Conc. (ng/ul) | Yields in 70ul (ng) | Volume for 150ng |
N37-1 | 4.48 | 313.30 | 33.51 |
N37-2 | 2.81 | 196.92 | 53.32 |
N37-3 | 4.33 | 302.97 | 34.66 |
N37-4 | 4.41 | 308.72 | 34.01 |
N37-5 | 4.04 | 282.85 | 37.12 |
N37-6 | 5.03 | 351.84 | 29.84 |
N37-7 | 4.74 | 332.11 | 31.62 |
N37-8 | 3.99 | 279.46 | 37.57 |
N37-9 | 4.11 | 287.69 | 36.50 |
N37-10 | 4.54 | 317.72 | 33.05 |
- Combine 150ng of each library - Perform PAGE size-selection of the size range 400-600bp in 6% TBE 5-well gel (2 gels) File:ZhangLab 2 2012-08-27 19hr 11min PAGE-SS WGBS N37.png - Resuspend with H2O, total volume 45ul - Quantified concentration by Qubit dsDNA assay BR (based on size 375bp): nM or XXng/ul - Concentration by PAGE quantification is XXng/ul
- Library IDs: NP_WGBS_N37_Aug26, 2012, sequenced on XXX run at XXX (Lane : XXX)