IGFBP6 knokdown in MDA-MB-231 cell line

Breast cancer line MDA-MB-231 was cultured in complete culture medium containing high glucose DMEM (4.5 g/L) supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 1% antibiotic-antimycotic solution ( Gibco) in an MCO-18AC incubator (Sanyo, Japan) at 37ºC and 5% CO2 concentration in the air. Cell reseeding was carried out every 2-3 days according to the standard protocol. The protocol for obtaining the MDA-MB-231 cell line with stable knockdown of IGFBP6 was described previously (Nikulin et al., 2021). Overall design: The RNA extraction was performed using the miRNeasy Micro Kit (Qiagen, Germany). RNA samples were treated for ribosomal RNA removal using the MGIEasy rRNA Depletion Kit (MGI, China), and the purified RNA library was prepared for NGS using MGIEasy Directional Library Prep Set (MGI, China). Libraries were sequenced on the DNBSEQ-G50 sequencing platform (MGI, China) with High-throughput Sequencing Set (FCL, SE100).