GSM8669512: p1492s3_Prol_SRSF7_H2O2_Papantonis; Homo sapiens; OTHER
Total RNA was isolated with the DirectZol RNA mini-prep kit (ZymoReaserch, R2052). The extracted RNA was afterwards used in Streptavidin pull-downs for enrichment of biotinylated RNA content. Briefly, 40 µl of Dynabeads_MyOne_Streptavidin C1 magnetic beads (Thermo Fisher, 65002) were prepared per 100 µg of RNA according to manufacturer's instructions. The beads were incubated with the extracted RNA in 1M NaCl, 5 mM  Tris-HCl pH 7.5, 0.5 mM EDTA and 25 Units/ml RiboLock RNase Inhibitor for 15 min at room temperature under gentle rotation. Finally, the enriched RNA was eluted form the beads with Trizol, purified with the RNA Clean & Concentrator-5 (ZymoResearch, R1014) according to manufacturer's instructions and quantified with the Qubit™ RNA High Sensitivity (HS) Assay Kit (Thermo Fisher, Q3285). Library preparation was performed using the SMART-Seq Total RNA Pico Input with UMIs kit (ZapR Mammalian; TAKARA Bio; Cat. N° 634354). This kit is specifically designed to efficiently prepare Illumina sequencing libraries from minimal input amounts (250 pg–10 ng) of total RNA, including RNA of high quality, partially degraded, or low quality. The resulting libraries retain strand information and incorporate indexes and adapters during the reverse-transcription and PCR amplification steps. Unique molecular identifiers (UMIs) are introduced during the reverse-transcription step to reduce potential PCR bias and provide additional precision for transcript quantification.