Noi/NOTES/2012-7-27
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WGBS sequencing library preparation using KAPA protocol (Eppendorf)
- Note that I used the same total volume of reaction similar to the protocol from Eppendorf. This might be slightly different from the original protocol from KAPA kit, but the total amount of enzyme and buffer are the same. I also adjust the volume of adapters in ligation step, but total volume and ratio of adapter to A-tailed DNA are the same.
Preparation of enzyme mix for End repair, A-tailing, Ligation, and Methylation adapters
- First of all, need to check if I have enough reagents for 8 rxn
- Since I perform library prep for exactly 8 rxn (1 column-> single pipetting), I will not prepare extra volume since I plane to set up machine to pipette all reaction mix aliquoted in the well (exactly each reaction need). This mean setting up larger volume of pipetting to make sure that all reaction mix is transfered to the DNA.
End repair Total reaction in 70ul DNA 50ul End repair reaction mix 20ul KAPA End repair mix 1x rxn 8x rxn mix 10X End repai buffer 7.00 56.00 KAPA End repair enzyme 5.00 40.00 H2O 8.00 64.00 ================================================== Total 20.00 160.00 Aliquot to column #1: 20ul 20C for 30min
A-tailing Total reaction in 30ul A-tailing reaction mix 1x rxn 8x rxn mix KAPA 10X A-tailing buffer 3.00 24.00 KAPA A-tailing enzyme 3.00 24.00 H2O 24.00 192.00 ================================================== Total 30.00 240.00 Aliquot to column #2: 30ul 30C for 30min
Adapter ligation Total reaction in 50ul Ligation reaction mix 40ul Methylation adapter 10ul Ligation reaction mix 1x rxn 8x rxn mix KAPA 5X Ligation buffer 10.00 80.00 KAPA DNA ligase 5.00 40.00 H2O 25.00 200.00 ================================================== Total 40.00 320.00 Aliquot to column #3: 40ul Methylation adapter preparation Alan's note: 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 1ug of DNA, used 2ul of adapters, but dilute to final volume 10ul (This volume is larger than KAPA protocol, but the total amount is the same) Methylation adapter 2ulx 8rxn 16.00 H2O 8ulx8rxn 64.00 ================================================== Total 80.00 25C for 15min