GSM2337061: COLO858_vem_24h_rep2; Homo sapiens; RNA-Seq
At the time of harvest for RNA, cells were washed once with PBS, then lysed in the dish with RLT buffer (Qiagen). Samples were immediately processed with Qiashredder and RNeasy kits (Qiagen) and frozen until further use. 10 μg of RNA was DNase-treated and cleaned up with RNeasy MinElute kit (Qiagen). RNA quality was assessed by Bioanalyzer (Agilent) and all samples had RINs of 9.0 or higher. RNA-seq libraries were constructed using Illumina’s TruSeq stranded mRNA library prep kit and protocol with minor modifications. Briefly, 1 μg of RNA was mixed with 2 μl of a 1:100 dilution of ERCC spike in control Mix2 (Life Technologies) before mRNA purification. Elution and fragmentation was done for 6 min at 94oC. cDNA was synthesized and cleaned up with Ampure beads (Agencourt). Fragments were end-modified and adaptors ligated before another Ampure bead cleanup. Final library amplification was done at 13 cycles and again cleaned up with Ampure beads. The resulting libraries were roughly 380 bp in length as assessed by Bioanalyzer. RAN-seq was performed at the Harvard University Sequencing Facility (FAS Division of Science) on Illumina HiSeq 2000 machines using the standard single-read (1 × 50 bp) protocol.
GEO Accession
GSM2337061