RNAseq of Homo sapiens: triple negative breast cancer from FUSCCTNBC project
RNA library preparation was performed as described in the Illumina TruSeq Stranded Total RNA LT sample preparation kit with Ribo-Zero Gold (Illumina Inc., San Diego, CA, USA). Briefly, ribosomal RNA was depleted from 0.3-1 g of total RNA. Following depletion, the mRNA was fragmented to an average insert size of 200400 bp at 94C for 4 min. The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase (Invitrogen) and random primers. The first-strand cDNA was converted into double-stranded DNA in the presence of dUTP. The incorporation of dUTP in second-strand cDNA synthesis quenches the second strand during amplification, thus improving the strand specificity of the library. These cDNA fragments were subjected to the addition of a single 'A' base and subsequent ligation of the adapter. The products were purified and enriched via PCR to generate the final library.