Sperm methylome of Murrah bull
The Reduced Representation Bisulfite Sequencing RRBS libraries were prepared using ZymoSeq RRBSLibrary Kit Catalog D5461 Zymo Research In brief 300ng of genomic DNA was used to get the CpG richgenomic fragments with the help of restriction enzyme MspI 5CCGG by incubating at 37C for 4 hrs Furtherthe CpG rich genomic fragments were ligated with linker adapters and gap filling was performed with 5Methylcytosine dNTP mix The ligated fragments were purified with Zymo spin IC column Later on the DNAfragments were treated with lighting conversion reagent which can chemically modify the non methylatedcytosine into uracil upon the addition with L desulphonation bufferFurthermore the Index primer amplification was done using standard dNTPs and an uraciltolerant Taq DNApolymerase which generates libraries with thymines in place of the originally unmethylated cytosines Theamplified product was then purified using Zymo spin IC column and the final library was eluted in 20l of DNAelution bufferLibrary QuantificationThe library concentration was determined in a Qubit3 Fluorometer Catalog Q33216 Life technologies usingThe Qubit dsDNA HS High Sensitivity Assay Kit Catalog Q32854 ThermoFisher Scientific The Qubit1X dsDNA HS Assay Kit DNA HS Assay Kit contains High Sensitivity DNA reagent as well as buffers and twoDNA standards DNA HS assay reagent is one of the most sensitive detection dye for the accurate quantitationof DNAlibrary in solution with linear fluorescence detection in the range of 10pgl to 100ngl of DNADye and the buffers were diluted at 1200 ratio and 1l of the library was mixed with the dye mix and incubatedat RT for 2min and the readings were taken in the Qubit3 Fluorometer Catalog Q33216 Life technologiesPrior to the samples measurement the instrument was calibrated using the two standards provided in the kit