Kapa stranded mrna seq platforms kit

Paired-end sequencing (2 x 50 bp) of total RNA was carried out at the Centre Nacional de Anàlisi Genòmica (CNAG). Sequencing methods have been reported by Guan et al. (2020) (link). Briefly, the RNA-Seq library was prepared with the KAPA Stranded mRNA-Seq Illumina Platforms Kit (Roche, Sant Cugat, Spain) by using 500 ng total RNA as template. Oligo-dT magnetic beads were used to enrich the poly-A fraction and subsequently, RNA was fragmented. Strand cDNA synthesis was performed in the presence of dUTP to enforce strand-specificity. The blunt-ended double stranded cDNA was 3′-adenylated and ligated to Illumina adaptors with unique dual indexes and unique molecular identifiers (Integrated DNA Technologies, Coralville, IA). Enrichment of the ligation product was ensured by performing 15 cycles of polymerase chain reaction amplification. An Agilent 2100 Bioanalyzer equipment was employed to verify the quality of the final library by using the DNA 7500 assay (Agilent Technologies, Inc., Santa Clara, CA). Library sequencing was carried out with a HiSeq 4000 equipment (Illumina, San Diego, CA) in accordance with the protocol for dual indexing advised by the manufacturer. Image analysis, base calling and quality scoring of the sequencing run were checked with the Real-Time Analysis (RTA 2.7.7) tool (Illumina, San Diego, CA) and FASTQ sequence files were subsequently generated.