3' mRNA sequencing of MCF7 cells upon E2 induction

After hormone deprivation (HD) for 72 hours, MCF7 cells were treated with 100 nM 17 b-Estradiol (E2) for 45 minutes, 3 hours, and 12 hours. Total RNA was isolated. The RNA integrity (RIN) values of samples were determined using the Eukaryote total RNA 6000 Nano kit and 2100 Bioanalyzer (Agilent Technologies, USA). All the samples used had RIN scores exceeding 7, indicating that the RNA samples were good quality, intact, and reliable for downstream analysis. Library generation and amplification steps for each sample were done using QuantSeq 3' mRNA-Seq library prep kit for ion torrent according to the instructions provided precisely (Lexogen, CAT012.24A). Libraries were run on the Agilent Bioanalyzer with an Agilent RNA 6000 Nano Kit chip to assess quality. The prepared 3'seq libraries were sequenced using the IonXpress System kit (ThermoFisher) on the Ion GeneStudio S5 TM (ThermoFisher). We used the IonXpress System (Illumina, San Diego, CA, USA) single-end run mode for sequencing.