Illumina HiSeq 4000 sequencing; RNA-seq of hESCs overexpressing inducible KLF17 versus uninduced controls.
RNA-seq of hESCs overexpressing inducible KLF17 versus uninduced controls.
RNA was isolated using TRI reagent (Sigma) by applying 1 ml reagent in-plate to one 6-well of cells and scraping to collect all material. Doxycycline-inducible overexpression of HA-tagged proteins was achieved using the Lenti-X Tet-On 3G Inducible Expression System (Clontech) following the manufacturer's protocol, and as outlined previously (Wamaitha et al., 2015). Lentiviral packaging was achieved using 7 μg of transgene-containing plasmid and the Lenti-X Packaging Single Shot reagents. Lentiviral supernatant was harvested after 48hrs and concentrated by ultracentrifugation. To produce stably transduced cells, hESCs were plated under standard conditions and changed into fresh medium the following morning. 24hrs post-plating, 10 μl concentrated virus was added to hESCs for transduction overnight (~16hrs). hESCs were dual selected with 150 μg/ml G418 and 0.5 μg/ml puromycin 48hrs post-transduction. For the RNA-seq experiments, KLF17-inducible hESCs were plated as normal and induction initiated after 24 hours by addition of 1 μg/ml Dox to the culture medium (mTeSR1). At ~30 hours, a day 0 (pre-induction) control sample was collected, then both induced (+Dox) and uninduced (UI) samples were collected at 24hr intervals from 48hrs (day 1 post-induction) until 144hrs (day 5 post-induction). TRI reagent samples were subjected to phenol/chloroform-based extraction, and DNase I-treated (Ambion). cDNA was synthesised using a Maxima first strand cDNA synthesis kit (Fermentas). DNase-treated RNA was supplied to the in-house sequencing facility and subjected to library construction via the KAPA mRNA (polyA) HyperPrep protocol.