RNA-Seq of Pongo pygmaeus: Jago, KD2
B-Lymphoblastoid cell lines immortalized by Epstein-Barr virus (EBV) of three individuals in each primate genus were cultured in RPMI 1640 media [Sigma Aldrich, cat: R0883] supplemented with 10% fetal bovine serum [Sigma Aldrich, cat: F1051], 2 mM L-glutamine [Sigma Aldrich, cat: G7513], and 5000 U/ml Pen/Strep [Sigma Aldrich, cat: P4333] at 37C and 5% CO2. siRNA transfection was performed following the DharmaFECT 1 protocol. Transfection was conducted with either a negative control siRNA or one of three different siRNAs designed for targeting ZEB2. Untreated cells served as an additional control. The two best performing siRNAs and the time point of 6 hours were chosen for knocking-down ZEB2 expression in all B-cells. Total RNA was extracted 6 hours after transfection from untransfected cells, the negative control and the two ZEB2 KD samples. For RNA-Seq, 500 ng of total RNA underwent library synthesis using TruSeq RNA Sample Prep Kit v2 [Illumina, cat: RS-122-2001] following the manufacturers protocol. The resulting barcoded libraries were cleaned up and quantified with the KAPA Library Quantification Kits - Illumina/Universal [Roche, cat: KK4824] on a TaqMan 7500 Real Time PCR System. A pool comprising up to 10 libraries was used for cluster generation at a concentration of 10 nM on an Illumina cBot. Sequencing of paired-end 100 bp fragments was performed using an Illumina HighScan-SQ sequencer at the sequencing core facility of the IZKF Leipzig (Faculty of Medicine, University Leipzig) employing version 3 chemistry and flow cell according to the manufacturers instructions.