MDAMB231-sh22_p
MCF7 and MDA-MB-231 cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), and 1% penicillin/streptomycin (Gibco). For MCF7 and MDA-MB-231, media was additionally supplemented with 10 µg/ml human insulin (Sigma-Aldrich). MCF10-A cell line was cultured in DMEM/F12 (Sigma-Aldrich) supplemented with 5% heat-inactivated horse serum (Gibco), 20 ng/ml epidermal growth factor (Sigma-Aldrich), 0.5 mg/ml hydrocortisone (Sigma-Aldrich), 100 ng/ml cholera toxin (Sigma-Aldrich), 10 µg/ml insulin (Sigma-Aldrich), and 1% penicillin/streptomycin (Gibco). Cells were cultured at 37°C in a humidified incubator at 5% CO2. HEK293T cells were co-transfected with pLKO.1-Puro containing shRNA1 and shRNA2 against METTL3 or scramble control (Table S1), the packaging vector pCMV-dR8.2-dvpr and the envelope vector pCMV-VSV-G (ratio 6:8:2), with Jet-PEI Polyplus following the manufacturer's instructions. Lentiviral particles were collected after 48 and 72 hours, filtered through a 0.45 µm filter and concentrated using Amicon Ultra-15 Centrifugal Filter (Merck). Knockdown of METTL3 was obtained by lentiviral transduction with the lentiviral particles in media supplemented with Polybrene (8 µg/ml). Transduced cells were selected by supplementing the culture media with puromycin (1 µg/ml) for an additional 4 days. Total RNA was extracted using the RNeasy Mini Kit (Qiagen) following the manufacturer's recommendations. 1 ug of RNA from each sample was used for the following protocols. Paired-end libraries were built using Illumina Tru-seq RNA protocols by the Novogene company.