RDhi10530_GCCAAT.experiment
The libraries are prepared in accordance with the manufacturer's protocol using Illumina TruSeq Stranded Total RNA Library Prep Kit. The first step involves the removal of ribosomal RNA (rRNA) from total RNA using biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads. The Ribo-Zero Human/Mouse/Rat kit depletes samples of cytoplasmic rRNA. Following purification, the RNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then have the addition of a single 'A' base and subsequent ligation of the adapter. The products are purified and enriched with PCR to create the final cDNA library.