title: 6C1
organism: Homo sapiens
description: Protocols: Colo-205 and RKO cells were were obtained from the American Type Culture Collection (ATCC) and 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. Cells were seeded in 6-well plates and left to attach overnight. Treatment with the indicated compounds was performed for 24h. RNA was extracted from cells using the TRIzol Reagent (Invitrogen) followed by a clean-up step combined with on-column DNase digestion using the RNeasy kit (Qiagen) according to manufacturers' instructions. 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.