GSM7295182: EO771_BrM3_MVC15 Brain metastasis-associated microenviroment; Mus musculus; RNA-Seq
RNA was harvested using Rneasy kit (Qiagen). 1ug of total RNA was used for the construction of sequencing libraries. Total RNA samples [1µg] were converted into sequencing libraries with the "NEBNext Ultra II Directional RNA Library Prep Kit for Illumina" (NEB #E7760), as recommended by the manufacturer. Briefly, polyA+ fraction is purified and randomly fragmented, converted to double stranded cDNA and processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters. Adapter-ligated library is completed by PCR with Illumina PE primers. {This kit generates directional libraries stranded in the antisense orientation [the read1 (the only read in single read format) has the antisense orientation (--library-type fr-firststrand in TopHat, --stranded=reverse in HTSeq]} The resulting purified cDNA libraries were applied to an Illumina flow cell for cluster generation and sequenced on an Illumina instrument by following manufacturer's protocols.