Homo sapiens
Protocols: Cervix adenocarcinoma cell line (HeLa), human triple-negative breast cancer (TNBC) model cell line (MDA-MB-231) and pleural effusion of hormone-dependent human breast carcinoma (MCF-7) were obtained the American Type Culture Collection (ATCC). HeLa and MBA-MD 231 were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA). MCF7 cell line was cultured in Dulbecco&#39;s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA). All media were supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine. Cells were maintained at 37 °C in a humidified incubator with 5% CO₂. Total RNA was extracted from frozen cell samples using Qiagen RNeasy Plus Universal mini kit following manufacturer's instructions (Qiagen, Hilden, Germany).     RNA samples were quantified using Qubit 3.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) and RNA integrity was checked with 4200 TapeStation (Agilent Technologies, Palo Alto, CA, USA). The RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina using manufacturer's instructions (New England Biolabs, Ipswich, MA, USA). Briefly, mRNAs were initially enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 3.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).