title: GSM4459419: PCX-OE_4; Homo sapiens; RNA-Seq
library_construction_protocol: Ishikawa cells were cultured overnight at 5.6 x 105 cells/well in a 6-well plate in MEM medium supplemented with 10% FBS, 1% antibiotic-antimycotic and 1% L-glutamine. The following day, cells were washed with PBS and total RNA was isolated from control and PCX-overexpressing Ishikawa cells using the RNeasy Mini Kit (Qiagen), and treated with TURBO DNA-free kit (Invitrogen). Total RNA was converted to strand specific Illumina compatible sequencing libraries using the NEXTflex Rapid Directional mRNA-Seq library kit from BIOO Scientific (Austin ,Texas) as per the manufacturers instructions (v14.10). 400ng of total RNA was polyA-selected and the mRNA fragmented prior to reverse transcription and second strand cDNA synthesis using dUTP. The resultant cDNA is poly adenylated before the ligation of Illumina-compatible barcoded sequencing adapters. The cDNA libraries were treated with UDG to degrade the second strand and PCR amplified for 14 cycles prior to assessment by Agilent Bioanalyzer for quality and Qubit fluorescence assay for amount.  Sequencing pools were generated by mixing equimolar amounts of compatible sample libraries based on the Qubit measurements.  Sequencing of the library pools was done with an Illumina Nextseq 500 using single read 75bp v2 sequencing chemistry