Dynabead mrna direct micro purification kit

Cells were seeded on 10 cm plates 2 days before doxycycline addition. After 24 h of doxycycline treatment, cells were lysed in RNA Extracol (EurX, Gdańsk, Poland). Total RNA was isolated using the Direct-zol RNA Mini (Zymo Research, Irvine, CA, USA); the procedure included on-column DNase I digestion. Poly(A)⁺ fractions were isolated using the Dynabead mRNA DIRECT Micro Purification Kit (Thermo Scientific) according to the manufacturer’s recommendations with minor modifications, such as three (instead of two) rounds of RNA binding, washing, and elution and an additional wash in detergent-free buffer B before the final elution step. Library preparations and sequencing on an Ion Torrent platform were performed at the Genomics Centre at the Malopolska Centre of Biotechnology (Kraków, Poland). The RNA-seq data presented in this article have been deposited in the GEO database (GEO: GSE178458). Reads per million mapped read values of samples treated with doxycycline and untreated were compared, and the transcripts that were downregulated in both cell lines were subjected to analysis in g:Profiler (https://biit.cs.ut.ee/gprofiler/gost).⁶⁰ (link) Based on g:Profiler analysis and published results, transcripts encoding proteins involved in the regulation of the cell-cycle progression and cell division were selected for further analysis.