GSM8197261: HUVECs, PPCN-RGD, 72h, rep 1; Homo sapiens; RNA-Seq
Extraction of RNA was carried out utilizing the Aurum™ Total RNA Mini Kit (Bio-rad), following the manufacturer's protocol. The quality of the extracted RNA was assessed using the Agilent Bioanalyzer 2100, with acceptance criteria for RNA integrity numbers (RIN) set at > 7. Quantification was performed using Qubit. To construct RNA sequencing libraries, Illumina TruSeq mRNA Sample Preparation Kits were employed, adhering to the manufacturer's instructions. The procedure involved the capture of polyadenylated mRNAs from total RNA through oligo-dT selection. Subsequently, cDNA synthesis occurred through reverse transcription, and each sample was ligated to Illumina sequencing adapters featuring unique barcode sequences. Following this, barcoded samples underwent PCR amplification, and the resulting cDNA libraries were quantified using qPCR. To achieve a comprehensive overview, equimolar concentrations of each cDNA library were pooled and sequenced on the Illumina HiSeq 4000. Subsequently, the quality of reads in FASTQ format was evaluated using FastQC. Trimming of reads, specifically removing Illumina adapters from the 3' ends, was executed using cutadapt. Trimmed reads were aligned to the human genome (hg38) using STAR. Read counts per gene were computed utilizing htseq-count alongside a gene annotation file for hg38 obtained from Ensembl. Normalization and differential expression analyses were conducted using DESeq2 1.42.0, employing the Wald test. The criterion for identifying significantly differentially expressed genes was an FDR-adjusted p-value less than 0.05, employing the Benjamini-Hochberg method. Genes were annotated with biomaRt 2.58.0 using GRCh38.p14 human genome. Gene set enrichment analysis was done using clusterProfiler 4.10.0 with gene sets c2.all.v2023.2.Hs.symbols, h.all.v2023.2.Hs.symbols, and c5.all.v2023.2.Hs.symbols. To construct RNA sequencing libraries, Illumina TruSeq mRNA Sample Preparation Kits were employed, adhering to the manufacturer's instructions.