title: GSM9530314: PBMC, Group 1, Baseline, rep 3; Macaca mulatta; RNA-Seq
library_construction_protocol: Blood collected in EDTA tubes (Sarstedt) was processed immediately. PBMCs were isolated by density gradient centrifugation (Lymphocyte Separation Medium, MP Biomedicals) at 1500 rpm for 45 min for phenotyping and functional assays. Tissues were collected in 1 mL Trizol reagent (15596026; Invitrogen) and extracted with RNeasy Mini Kit (Cat. No.74104; QIAGEN, Hilden, Germany) following the manufacturer's protocol. The concentration of RNA was determined by NanoDrop 2000. Final cDNA libraries carrying TruSeq RNA CD indexes (Illumina, 20019792) were quantified with the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Q32854). Library quality was assessed on an Agilent TapeStation 4150 using Agilent D1000 ScreenTape (Agilent, 5067-5582). Smear analysis (Agilent TapeStation Software, Version 4.1.1) with a 600 bp window was used to determine the average library size. Library molarity was then calculated from the measured size and concentration. Libraries were pooled to a final concentration of 750pM with a 2% spike-in of PhiX control library v3 (Illumina, FC-110-3001). The pooled mixture was loaded onto an Illumina NextSeq P1 (300) reagent cartridge (Illumina, 20050264). Paired-end, dual-index sequencing (150×8×8×150) was run on the NextSeq2000, generating ~20 M paired-end reads per sample. Fastq files produced by Illumina BaseSpace DRAGEN Analysis Software (Version 1.2.1) were used for downstream analyses.