RNAseq of HK-2 human kidney tubular cells knock-out for CNDP2
RNAseq of HK-2 human kidney tubular cells knock-out for CNDP2
Immortalized human tubular cells (HK-2; American Type Culture Collection CRL-2190) were grown in RPMI 1640 GlutaMAX medium (Thermo Fisher Scientific. Waltham. MA) with 0.1 or 10 % fetal calf serum (v/v) and 1% penicillin and streptomycin (v/v) at 37°C with 5% CO2.  A gRNA targeting exon4 of the human CNDP2 locus was designed using CRISPOR (PMID: 27380939). The corresponding DNA sequence was cloned into the plasmid px549 pSpCas9(BB)-2A-Puro (Addgene plasmid #48139) and transfected into HK-2 cells. After 5 days of Puromycin (3 μg/ml) selection, clones were isolated and the genomic DNA was subjected to PCR amplification of a 450bp amplicon centered on the targeting site. PCR product was sent to sequence and positive clones were validated in western blot for CNDP2 protein expression. Total RNA was extracted using the peqGOLD total RNA kit (VWR, Darmstadt, Germany) including an in column treatment with RNAse-free DNAseI (Qiagen) to eliminate potential genomic DNA contaminants. mRNA-Seq libraries were created according to the Truseq stranded mRNA Illumina protocol.