Noi/NOTES/2012-8-26

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WGBS library preparation of low input DNA N37 10 tissues samples using KAPA protocol (from Eppendorf) and epMotion[edit]

Preparation of enzyme mix for End repair, A-tailing, Ligation, and Methylation adapters[edit]

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[edit]

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[edit]

  • 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 (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[edit]

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

1. Use the methylated adapter with paired-end PCR primer pairs(PCR primer PE1.0 and PCR primer PE 2.0) provided in illumina's paired end genomic DNA sample prep kit
2. Use 10ul of methylated adapter to 5ug gDNA insert, if starting DNA amount is less than 5ug, titrate the volume of adapter reagent accordingly to maintain 10:1 ratio of DNA
Since I used 0.2ug of DNA, used 0.2ul of adapters, but dilute to final volume 10ul (This volume is larger than KAPA protocol, but the total amount is the same)
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[edit]

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[edit]

  • Performed bisulfite conversion manually
  • Elute with 40ul Elution buffer (final volume ~35ul)

Amplification of bisulfite sequencing libraries with N2 barcoded primers[edit]

  • 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.Ind1-8,21,22 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[edit]

 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[edit]

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)