GSM6914453: Mammary adenocarcinoma, database ID 21-001-01 [HET.30]; Mus musculus; RNA-Seq
Total RNA was isolated from tumour tissue using the RNeasy Minikit (QIAGEN) according to the manufacturer's protocol. Samples for RNAseq analysis underwent an on-column DNase I digestion step for genomic DNA removal prior to further processing. Total RNA quality and quantity was assessed using Agilent 4200 TapeStation and hsRNA or RNA ScreenTapes (Agilent Technologies). mRNA was isolated from 50ng of total RNA (RIN value >7) using the NEBNext® Poly(A) mRNA magnetic isolation module, (New England BioLabs, NEB, #E7490) and the sequencing libraries were prepared using the NEB® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB, #E7760). The sequencing libraries were prepared following Chapter 1 of the NEB® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England BioLabs, NEB) protocol. The steps included mRNA isolation, fragmentation and priming, first strand cDNA synthesis, second strand cDNA synthesis, adenylation of 3' ends, adapter ligation (1:80 dilution) and PCR amplification (14-cycles). Libraries were validated using the Agilent 4200 TapeStation and hsD1000 ScreenTapes (Agilent Technologies) to ascertain the insert size, and the Qubit® (Thermo Fisher Scientific) was used to perform fluorometric quantitation. The manufacturer's instructions were followed except for the replacement of SPRIselect Beads or NEBNext Sample Purification Beads by AMPure XP beads (Beckman Coulter®) in purification steps. The validated libraries were normalized to 4nM, pooled together and the pool sequenced on an S1 (200 cycle) flow cell using a 2x100bp PE dual index format on the Illumina® NovaSeq6000 sequencing system according to the manufacturer's instructions. Samples were sequenced to a read depth of at least 35 Million prior to quality trimming with fastp