title: RNAseq of MV4-11 AML cells of control versus SKI knock-out treated either with dimethyl sulfoxide (DMSO) or phorbol myristate acetate (PMA)
design: RNAseq of MV4-11 AML cells of control versus SKI knock-out treated either with dimethyl sulfoxide (DMSO) or phorbol myristate acetate (PMA)
library_name: SKI KO-3_III_DMSO_s
library_construction_protocol: RNA for RNA-sequencing (RNA-seq) was extracted from MV4-11 CTR and SKI KO clones treated with 1.5 ng/ml PMA or DMSO solvent control for 24 h. Generation of MV4-11 CRISPR/Cas9-mediated SKI knockout single cell clones (SKI KO) was performed as described in Feld et al. Nucleic Acids Res. 2018; 46(7) 3412–3428. A sgRNA against GFP was used as control (CTR). MV4-11 clones were cultured in RPMI (Life Technologies, Carlsbad, USA) supplemented with 10% FCS (Capricorn Scientific; Ebsdorfergrund, Germany) and 1% Penicillin/Streptomycin (P/S) (PAA Laboratories; Pasching, Austria). Selection and maintenance of lentiviral transduced cells was performed in the presence of 0.5 µg/ml puromycin. MV4-11 CTR and SKI KO clones were treated with 1.5 ng/ml PMA or DMSO solvent control for 24 h prior to harvesting. RNA was isolated using the RNeasy Mini kit (Qiagen) and RNA quality was assessed using the Experion RNA StdSens Analysis Kit (Bio‐Rad).  RNAseq libraries were prepared from total RNA with the Lexogen QuantSeq 3'-mRNA Library Prep Kit FWD for Illumina (Lexogen, 015.24) in combination with the UMI Second Strand Synthesis Module for QuantSeq FWD (Illumina, Read 1) (Lexogen, 081.96) following the manufacturer's protocol. Quality of sequencing libraries was controlled on a Bioanalyzer 2100 using the Agilent High Sensitivity DNA Kit (Agilent).