title: GSM9145990: 4T1_KIRA6_Rep1; Mus musculus; RNA-Seq
library_construction_protocol: RNA was havested using TRIzol (Invitrogen) according to manufacturer's' instructions. 1 μg total RNA was used for library preparation. For mRNA enrichment, poly(A) mRNA isolation was performed using Oligo(dT) beads. The mRNA fragmentation was performed using divalent cations and high temperature. Priming was performed using Random Primers. First strand cDNA synthesis was carried out using reverse transcriptase, followed by second-strand cDNA synthesis. For strand-specific library construction, during the second-strand synthesis the standard dTTP was replaced with dUTP; subsequent treatment with USER enzyme selectively degrades the second strand prior to PCR amplification, thereby preserving strand information. The purified double-stranded cDNA was then treated to repair both ends and add a dA-tail in a single reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of adaptor-ligated DNA was then performed using DNA Clean Beads. Each sample was then amplified by PCR using P5 and P7 primers, and the PCR products were validated.