title: GSM2593739: BR_MVA_WR_0_5d_M25_exp1; Mus musculus; RNA-Seq
library_construction_protocol: For whole organs, samples were lysed in Trizol and total RNA was extracted by phenol-chloroform extraction followed by purification with Dynabeads MyOne Silane beads (Thermoscientific). For sorted cell populations, CD8+ T cell populations (5,000-10,000 cells per sample) were lysed in RLT buffer (Qiagen) containing 1% beta-mercaptoethanol and total RNA was extracted with Dynabeads MyOne Silane beads. After bead-based enrichment, total RNA samples were purified by on-bead DNase I treatment followed by cleanup with 80% ethanol. RNA concentration was normalized across samples prior library construction. Total RNA was heat-fragmented to an average size of 500 bp fragments and reverse transcribed with barcoded oligo(dT) primers containing an Illumina-compatible adapter sequence. After RNA degradation, barcoded, single-stranded cDNA samples were pooled and flanked by an Illumina-compatible adapter sequence in their 3’ end using a ‘primer extension’ method with DNA Pol I, Large Klenow Fragment exo minus. cDNA pools flanked on both ends with illumina sequences were then amplified by PCR and sequenced on Illumina sequencers.
attributes.tag: GEO Accession
attributes.value: GSM2593739