Transcriptome profiling of 17β-estradiol effects

Bulk RNA-sequencing was applied to profile transcriptomic changes under 17β-estradiol treatment. BMEC-like cells were purified onto 6-well plates, and 24 h after subculture, 10 µM of 17β-estradiol or DMSO was prepared in EC medium lacking bFGF and RA and added to the cells. After 24 h, BMEC-like cells were washed once with DPBS, lifted using a cell lifter (Fisher Scientific #08–100-240), and collected via centrifugation. Resultant cell pellets were lysed in TRIzol reagent (Thermo Fisher Scientific #15,596,026) at room temperature for 10 min and stored at − 80 °C. Three biological replicates were collected per condition. Following the manufacturer’s guideline, RNA from each sample was purified using a Direct-zol RNA Miniprep kit (Zymo Research #R2051) simultaneously. Purified RNA samples were submitted to the Vanderbilt Technologies for Advanced Genomics core and sequenced on an Illumina NovaSeq6000. Raw sequencing reads were trimmed and aligned to human genome (GRCh38) with HISAT2 2.2.0 [42 (link)], and genomic features were obtained using the featureCounts function [43 (link)]. Differential gene expression and meta read counts were analyzed with edgeR [44 (link)]. Gene ontology enrichments analysis was performed on genes with p < 0.05 and fold change greater than 1.5 using WEB-based Gene SeT AnaLysis Toolkit (WebGestalt) [45 (link)].