title: GSM2310562: BPA5; Macaca mulatta; RNA-Seq
library_construction_protocol: The cells were harvested and lysed approximately 24 hrs after the last media change.  At harvest, all colonies in each well were cut and lifted, and divided into two microcentrifuge tubes, and centrifuged at 3,000 x g for three min and supernatant removed. Cells were resuspended in 1 mL DPBS with 10% BSA and 25 mM EDTA and centrifuged as above. Supernatant was removed and the remaining cell pellet was lysed in 100 ul PicoPureTM extraction buffer (Thermo Fisher Scientific, Life Technologies, Grand Island, NY). RNA was isolated following the PicoPureTM RNA Extraction kit manufacturer protocol, with DNase digestion to remove any contaminating DNA. 100 ng of each RNA sample were processed first using the Ovation RNA-Seq System v2 using  Ribo-SPIATM Technology (Nugen, San Carlos, CA). This was followed by fragmentation to an average of 300 bp using a Covaris-2 sonicator and a brief S1 nuclease digestion.  After purification, the cDNA was processed through the Ovation Ultralow DR Multiplex Systems 1-8 and 9-16 (NuGen) for end repair, barcoding and final library production. Barcoded libraries were pooled, loaded on flowcells and sequenced with Illumina HISeq 2500 in rapid run mode to generate 50 nt single end reads. The samples were loaded at 65% of optimal loading concentration, along with PhiX Control library (Illumina) added at 10% of loading concentration to increase read sequence complexity. 50bp single-end RNAseq
attributes.tag: GEO Accession
attributes.value: GSM2310562