Revision as of 15:48, 30 August 2012 by >Noi
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
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 (A & B: 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 (A & B: 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 (A & B: 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.Ind1-10,21,22 |
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
dsDNA Qubit HS quantification
Library IDs
|
Concentration in the Qubit
|
Unit
|
uL used
|
Dilution
|
Final conc. (ng/ul)
|
Ave. Conc. (ng/ul)
|
AL-exome_Aug21 1ul |
46.60 |
ng/mL |
1.00 |
200 |
9.32 |
9.76
|
AL-exome_Aug21 2ul |
102.00 |
ng/mL |
2.00 |
100 |
10.20 |
|
NP_WGBS_N37_Aug26 1ul |
18.70 |
ng/mL |
1.00 |
200 |
3.74 |
3.80
|
NP_WGBS_N37_Aug26 2ul |
38.50 |
ng/mL |
2.00 |
100 |
3.85 |
|
NP-BSPP-GL-AL_Ind9-29_Aug23 1ul |
72.50 |
ng/mL |
1.00 |
200 |
14.50 |
14.95
|
NP-BSPP-GL-AL_Ind9-29_Aug23 2ul |
154.00 |
ng/mL |
2.00 |
100 |
15.40 |
|
Library IDs
|
Ave. Conc. (ng/ul)
|
Size (bp)
|
Conc. (nM)
|
MW (g/mole)
|
AL-exome_Aug21 |
9.76 |
300 |
53.52 |
182,377.9
|
NP_WGBS_N37_Aug26 |
3.80 |
500 |
12.49 |
303,857.9
|
NP-BSPP-GL-AL_Ind9-29_Aug23 |
14.95 |
375 |
65.59 |
227,932.9
|
Jeff' libraries
Library IDs
|
Concentration in the Qubit
|
Unit
|
uL used
|
Dilution
|
Final conc. (ng/ul)
|
Size (bp)
|
Conc. (nM)
|
MW (g/mole)
|
JG-pool1 |
3.44 |
ng/mL |
5 |
40 |
0.14 |
450 |
0.50 |
273,487.9
|
JG-pool2 |
2.56 |
ng/mL |
5 |
40 |
0.10 |
450 |
0.37 |
273,487.9
|
- Quantified concentration by Qubit dsDNA assay HS (based on size 375bp): 3.80ng/ul pr 12.49nM
File:ZhangLab 2 2012-08-28 14hr 52min PQ NP WGBS N37.png
- Concentration by PAGE quantification is 14.8ng/ul
- Library IDs: NP_WGBS_N37_Aug26, 2012, sequenced on HiSeqAug28 run at Biogem (Lane : 4-6)