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University Medical Center Clinic for General, Visceral and Pediatric Surgery Tumor Epigentics

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Namespace:University Medical Center Clinic for General, Visceral and Pediatric Surgery Tumor Epigentics

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Protocols: Colorectal cancer cell lines were cultured in medium maintained at 37°C and 5% CO2. ARID1A knockout cells were obtained by CRISPR/Cas9 mediated gene editing. ARID1A knockdown cells were obtained by siRNA mediated knockdown. Total RNA was extracted 48 h post seeding/transfection. HCT116 cells were cultured in McCoy’s 5A medium (Gibco), DLD1 and COLO320DM cells were cultured in RPMI 1640 (Gibco). All media were supplemented with 10% FBS, 1% glutamine and 100 U/mL penicillin/streptomycin. Cells were maintained at 37°C and 5% CO2. For siRNA mediated knock down, the Lipofectamine RNAiMax transfection reagent (Invitrogen) was used according to manufacturer’s instructions. The cells were harvested after 48 h. CRISPR/Cas9 mediated gene editing was used to generate ARID1A knockout colorectal cancer cell lines. The guide RNAs were cloned into a pSpCas9(BB)-2A-GFP (Addgene) plasmid. 4 μg of plasmid was transfected into the cells by electroporation using a Lonza Nucleofector. 48 h after transfection the cells were sorted as 192 single cell clones by fluorescence assisted cell sorting based on GFP-positivity. The single cell clones were expanded and selected for ARID1A knockout. Total RNA was isolated in triplicates using the QIAzol® lysis reagent (Qiagen) according to manufacturer’s instructions. Libraries for poly(A) mRNA-sequencing were prepared using the Capture and Amplification by Tailing and Switching (CATS) RNA-seq kit (Diagenode) according to manufacturer’s protocol using 50 ng total RNA as the starting material.