GSM7109935: NK cells, unstimulated, 0.5 hours, rep 2 [S2]; Mus musculus; RNA-Seq
RNA was isolated using Dynabeads mRNA DIRECT Kit (Invitrogen), and then reverse transcribed using the AffinityScript Multiple Temperature cDNA Synthesis Kit (Agilent Technologies) to synthesize cDNA. After first strand synthesis, samples were pooled together based on ACTB qPCR values, and RNA-DNA hybrids degraded using consecutive acid-alkali treatment. A second sequencing linker (AGATCGGAAGAGCACACGTCTG) was ligated using T4 ligase (NEB) followed by SPRI clean-up. The mixtures were then PCR enriched for17 - 20cycles and SPRI purified to yield final strand specific RNA-seq libraries. Individual librarieswere then pooled into two final librarypools for sequencing based on ACTB qPCR values. Final library QCwas performed using the Agilent TapeStation system. Libraries were sequenced on HiSeq 2500 (Illumina) using 40 bp X 10 bp paired-end sequencing.