title: GSM1401672: HCC-1187; Homo sapiens; RNA-Seq
library_construction_protocol: The frozen breast tissue specimens were weighed, transferred to a 15 mL conical tube containing ceramic beads, and RLT Buffer  (Qiagen) plus 1% BME was added so that the tube contained 35 uL of buffer for each milligram of tissue.  The conical tubes containing tissue, ceramic beads and buffer were then shaken in a MP Biomedicals FastPrep machine until the tissue was visibly homogenized (90 seconds at 6.5 meters per second).  The homogenized tissue was stored at -80°C.  Total RNA was extracted from 5 million cultured cells or 350 uL of tissue homogenate (equivalent to 10 mg of tissue) using the Norgen Animal Tissue  RNA Purification Kit (Norgen Biotek Corporation). Cell lysate was treated with Proteinase K before it was applied to the column and on-column DNase  treatment was performed according to the manufacturer’s instructions. Total RNA was eluted from the columns and quantified using the Qubit RNA Assay Kit and the Qubit 2.0 fluorometer (Invitrogen). RNA-seq libraries for each sample were constructed from 250 ng total RNA using the polyA selection and transposase-based non-stranded library construction (Tn-RNA-seq) described previously (Gertz et al. Transposase mediated construction of RNA-seq libraries.2012. Genome Res 22 (1):134-141). RNA-seq libraries were barcoded during PCR using Nextera barcoded primers according to the manufacturer (Epicentre). The RNA-seq libraries were quantified using the Qubit dsDNA HS Assay Kit and the Qubit 2.0  fluorometer (Invitrogen) and three barcoded libraries were pooled in equimolar quantities for sequencing. The pooled libraries were sequenced on an Illumina HiSeq 2000 sequencing machine using paired-end 50 bp reads and a 6 bp index  read, and we obtained at least 50 million read pairs from each library.
identifiers.namespace: GEO
identifiers.id: GSM1401672