GSM9271531: A11L169-Grp3-Day0-24h_PID22718_AID5442_Batch1_Mrk_RNA_Seq_20Mar2023_1679340942963; Macaca mulatta; RNA-Seq
Peripheral blood mononuclear cells (PBMCs) were collected to profile mRNA and compare adjuvant effects on immune gene expression. Blood draws occurred at six time points: pre-dose, 1 day after each dose, and 7 days after each dose. Whole blood was preserved in PAXgene Blood RNA tubes, and total RNA was isolated using the PAXgene Blood RNA kit (PreAnalytix) following the manufacturer's instructions. RNA quantity and integrity were evaluated on an Agilent Bioanalyzer using the RNA6000 Nano Lab Chip (Agilent Technologies). For samples collected in PAXgene tubes, cells were pelleted by centrifugation at 10,000 g for 6 minutes, the supernatant was discarded, and pellets were washed in 1 mL of dimethylpyrocarbonate (DMPC)-treated water before a second centrifugation at 10,000 g for 6 minutes. For samples from other media, cells were pelleted at 2,000 g for 30 minutes and the supernatant removed. All cell pellets were disrupted by vortexing in the appropriate lysis buffer for the chosen extraction method. Total RNA was extracted from samples using standard extraction methods ensuring high RNA integrity. RNA sequencing libraries were prepared using the TruSeq Stranded Total RNA Ribo-Zero kit (Illumina) according to the manufacturer's instructions. This kit depletes ribosomal RNA to enrich for coding and non-coding transcripts, preserving strand specificity. Following RNA extraction and library preparation, the libraries were sequenced on an Illumina platform using a paired-end 50 base pair read format. Sequencing depth and multiplexing were adjusted according to experimental design to ensure sufficient coverage for downstream transcriptomic analyses.