S3
After the QC procedures, mRNA from eukaryotic organisms is enriched usingoligo(dT) beads. For prokaryotic samples, rRNA is removed using a specialized kitthat leaves the mRNA. The mRNA from either eukaryotic or prokaryotic sources isthen fragmented randomly in fragmentation buffer, followed by cDNA synthesisusing random hexamers and reverse transcriptase. After first-strand synthesis, acustom second-strand synthesis buffer (Illumina) is added with dNTPs, RNase H andEscherichia coli polymerase I to generate the second strand by nick-translation. Thefinal cDNA library is ready after a round of purification, terminal repair, A-tailing,ligation of sequencing adapters, size selection and PCR enrichment.
SUB9514105
S3