GSM9081764: hRMEC, PGE2, sample 3; Homo sapiens; RNA-Seq
Cells were washed with cold PBS, lysed, and total RNA was extracted using the RNeasy Mini Kit (Qiagen; Germantown, MD; #74106) according to the manufacturer's protocol. RNA samples were submitted to the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core laboratory (Nashville, TN) for RNA sequencing (RNA-Seq). Total RNA was quantified using a BioTek Synergy Multimode Plate reader and BioTek Gen5 2.09 software before being processed for RNA sequencing. RNA quality was determined using the Agilent Bioanalyzer 2100 Instrument (Agilent; Santa Clara, CA). RNA-Seq libraries were prepared using 200 ng of total RNA and the NEBNext rRNA Depletion Kit (New England Biolabs; Ipswich, MA; #E6310X) according to manufacturer's instructions. This kit employs an RNaseH-based method to deplete both cytoplasmic (5S rRNA, 5.8S rRNA, 18S rRNA, and 28S rRNA) and mitochondrial ribosomal RNA (12S rRNA and 16S rRNA) from total RNA preparations. The mRNA was enriched via poly-A-selection using oligoDT beads (New England Biolabs) and then the RNA was thermally fragmented and converted to cDNA. The cDNA was adenylated for adaptor ligation and PCR amplified. The libraries were sequenced using the NovaSeq 6000 (Illumina; San Diego, CA) with 150 bp paired end reads targeting 50 M reads per sample.