Control_2
Homo sapiens
Protocols: RKO human colorectal adenocarcinoma cell lines were obtained from the American Type Culture Collection (ATCC) All cell lines were grown in DMEM medium supplemented with 10% Fetal Bovine Serum and 1% penicillin/streptomycin and amino acids (all from Thermo Fisher Scientific, Waltham, MA, USA) at 37°C, 5% CO2 RKO cells were seeded in 6-well plates and left to attach overnight. Upon treatment, total RNA was extracted using the Trizol reagent (Ambion by Life Technologies, Foster City, CA, USA) After the total RNA extraction and DNase I treatment, magnetic beads with Oligo (dT) are used to isolate mRNA. Then cDNA is synthesized using the mRNA fragments as templates. Short fragments are purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short fragments are connected with adapters. The suitable fragments are selected for the PCR amplification as templates.