GSM9312424: K037; Homo sapiens; RNA-Seq
The preparation and subsequent sequencing (using the Illumina NextSeq 550 instrument) followed exactly the protocol described in the Illumina Stranded mRNA Prep Ligation Reference Guide. Quickly summarized, this involved 8 steps - (1) purifying and fragmenting the messenger RNA by capturing mRNAs with polyA tails using oligo(dT) magnetic beads and then fragmenting and priming the RNA , (2) synthesizing the first strand cDNA (complimentary DNA) by reverse transcribing the hexamer-primed RNA-fragments, (3) synthesizing the second strand cDNA to generate blunt-ended and doubled-stranded cDNA fragments, (4) adenylating 3-inch ends by adding an adenine nucleotide and a thymine nucleotide to the ends of the blunt fragments to prevent them from ligating to each other and for providing overhang for ligating the adaptors, (5) ligating anchors to the ends of the cDNA fragments in preparation for dual indexing, (6) cleaning up fragments with magnetic beads (Numbered steps continued from extraction protocol): (7) amplifying the library using PCR to selectively amplify fragments and add indexes and primer sequences for cluster generation, and (8) cleaning up the library using magnetic beads to purify the dual-indexed libraries. The final libraries were checked using the Agilent 2100 Bioanalyzer and DNA 1000 kit and were then diluted to the starting concentration for the NextSeq550 model. Each of our samples required four lanes on the flow cells that went into the sequencing machine and each lane was run twice.