GSM6593562: OE 2 (over expression 2) Tamas; Homo sapiens; RNA-Seq
Total RNA was extracted from frozen brain samples by using the Direct-zol™ RNA Kit (Zymo Research) following the manufacturer's instructions. 1µl of total RNA was loaded on Total RNA Pico Chip and measured for its integrity with Bionalyzer 2100 (Agillent Biotechnology). To concentrate the samples for the preparation of RNA seq libraries, the RNAs were precipitated at -80°C  overnight by solution of isopropanol, Na-Acetate and glycogen. 500 ng total RNA was used to prepare seq Libraries by Roche KAPA RNA HyperPrep with  Ryboerase Kit according to the manufacturer instructions. High throughput sequencing was run at Hiseq4000, with single indexed, paired end, 75bp mode. To deplete PGBD1, we first used a CRISPR/Cas9-mediated KO approach, however, no stable, proliferative KO line could be generated in either hESCs or neuroprogenitor cells (NPC). 300,000 human neuroprogenitor cells (NPC) were transfected by different pmiRNA constructs together with pSB100 plasmid encoding SB100 transposase in ratio 10:1. 3 days posttransfection the pmiRNA contained cells were FACS sorted based on mCherry marker gene intensity. The sorted single cells were cultured for additional three weeks then cells were FACS sorted repeatedly. Then PGBD1 protein level was determined by Western blotting from individual stable cell lines lysate