V157F_N3a_3
Homo sapiens
Protocols: Cells were detached using 0.05 % Trypsin-EDTA (Gibco, 15400054). The human colorectal carcinoma cell line HCT116 were obtained from the American Tissue Collection Center (ATCC, CCL-247) and cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, 41966-029) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich, S0615) and 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco, 15140122). Cell lines were cultivated in a humidified atmosphere at 37 °C and 5% CO2. Cells were treated either with 10 µM N3a or the corresponding volume of DMSO, as solvent control, for 36 h prior to RNA isolation. RNA isolation was done using the RNeasy Mini Kit according to manufacturer's protocol. RNA quality was evaluated using the Experion RNA StdSens Analysis Kit (Bio‐Rad, 700-7103). RNAseq libraries were prepared from total RNA with the QuantSeq 3′ mRNA-Seq 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 to manufacturer's protocol.