title: GSM2545248: SKBR3 rep4 (RNA-Seq); Homo sapiens; RNA-Seq
library_construction_protocol: The cells were collected at ~70-80% confluence.  RNA isolation for RT-qPCR and RNA-seq was performed using the RNeasy Mini Kit (Qiagen). Total RNA was isolated as described above.  The integrity of the RNA was assessed and verified using an Experion Automated Electrophoresis System (Bio-Rad) before mRNA-seq libraries were prepared using methods described previously (Zhong et al., 2011).  Briefly, polyA+ RNA was enriched using Dynabeads oligo(dT)25 (Invitrogen), heat fragmented, and reverse transcribed using random hexamers in the presence of dNTPs.  Second strand cDNA synthesis was performed with dNTPs, but replacing dTTP with dUTP.  After end-repair, dA-tailing, ligation to adaptors containing barcode sequences, and size selection using AMPure beads (Agencourt), the synthesized second-strand was digested using uracil DNA glycosylase (Enzymatics).  A final PCR reaction was performed using Phusion high-fidelity DNA polymerase (NEB).  After library quality control assessment using a Bioanalyzer (Agilent), the samples were subjected to 50 bp single-end sequencing using an Illumina HiSeq 2000 Sequencing System.  At least two biological replicates for each cell line were sequenced