GSM8841439: 8h-VEGFA-1; Homo sapiens; RNA-Seq
HCAECs were grown in 6 cm plates (Thermo Fisher Scientific , 3 × 105 cells per plate), stimulated for 8 hours, and lysed in RLT buffer (Qiagen). The New England Biolabs NEBNext Poly(A) mRNA Magnetic Isolation Module was used for mRNA enrichment from 500 ng total RNA per sample. cDNA libraries were generated following the New England Biolabs NEB Next Ultra II workflow with 8 cycles of PCR. A DNA purification step was introduced at the end of the procedure using 1.2× Agencourt AMPure XP beads (Beckman Coulter). Fragment length distribution was monitored with the Bioanalyzer High Sensitivity DNA Analysis kit (Agilent). Libraries were quantified using the Qubit dsDNA HS Assay (Thermo Fisher Scientific). Individually barcoded libraries were pooled, denatured, diluted to 1.8 pmol l–1, and sequenced with an Illumina NextSeq 550 High Output Flowcell Kit (75 cycles) (20024906; Illumina), using the following settings: 38 bp each as sequence reads 1 and 2; 8 bp each as index reads 1 and 2. Samples obtained an average sequencing depth of 27 million reads.