We used 10x Chromium Single Cell 3’ Reagent kits v3 Chemistry (10x Genomics, Cat# PN-1000075) for monkeys F and P. We reverse transcribed RNAs and generated libraries according to 10x Genomics protocol. Briefly, we generated Gel beads-in-emulsion (GEMs) after running through a 10x Genomics Chromium controller. We reverse transcribed mRNAs within GEMs in a Bio-Rad PCR machine (Cat# C1000). We barcoded cDNAs from individual cells with 10x Genomics Barcodes and barcoded different transcripts with unique molecular identifiers (UMIs). We purified cDNAs with Dynabeads (10x Genomics, Cat# 2000048) after breaking the emulsion with a recovery agent (10x Genomics, 220016). Then, we amplified cDNAs by PCR and purified them with SPRIselect reagent (Beckman Coulter, Cat# B23318). We analyzed the cDNA quantification and quality using Agilent Bioanalyzer 2100. We prepared libraries following fragmentation, end repair, A-tailing, adaptor ligation, and sample index PCR. We quantified the libraries by qPCR using a KAPA Library Quantification Kit (KAPA Biosystems, Cat# KK4824). We pooled together libraries from individual monkeys and loaded them onto NovaSeq S4 Flow Cell Chip. We sequenced samples from monkeys F and P to depths of 400,000 and 250,000 reads per nuclei, respectively.
