title: GSM6923560: Tag8: Monkey AC3, NP3, AC4; Macaca fascicularis; RNA-Seq
library_construction_protocol: The NP sample was gelatinous, and AC samples were minced into small pieces (2 mm) using a biopsy punch. Next, both samples were washed with preparation medium (RPMI-1640 [Nacalai Tesque] with 0.2% FBS [Gibco] and 10 mM HEPES) and transferred to a 6-well plate containing digestion medium (preparation medium with 0.2 mg/ml Liberase TM (Roche) and 2 kU/ml DNaseⅠ (Merck) within 2 h of sacrifice. The plate was then placed on a shaker and incubated for 3 h at 37 °C and 5% CO2. After dissociation, the samples were applied to a 70 μm cell strainer (BD Biosciences), followed by the next step of the cell barcoding process.   For cell surface labeling, we biotinylated cell surface proteins as described (M. Sugimoto, Y. Tada, S. Shichino, S. Koyamatsu, N. Tsumaki, K. Abe, Universal Surface Biotinylation: a simple, versatile and cost-effective sample multiplexing method for single-cell RNA-seq analysis, DNA Res. 29(3) (2022).). Cells were resuspended in 1 mL PBS supplemented with 1% FBS and 1 ng EZ-Link Sulfo-NHS-Biotin (Thermo Scientific) for 10 min at 4 °C, washed twice with PBS supplemented with 3% FBS, and then washed with Cell Staining Buffer (BioLegend). Next, the cells were stained with 0.6 μg/ml of Totalseq (A0951-A0954 [BioLegend]) for 20 min at 4°C for cellular barcoding, washed with Cell Staining Buffer, washed with PBS supplemented with 1% FBS, and resuspended in PBS supplemented with 10% FBS and 1 μM Sytox blue dead cell stain (Invitrogen) for 5 min at room temperature.  Barcoded live dissociated cells (PE-positive and Sytox blue-negative) were sorted and suspended in PBS supplemented with 20% FBS. The data was collected on a FACS Aria II flow cytometer (BD Bioscience) and analyzed with BD FACS Diva 9.0.1 (BD Biosciences). Library preparation and the first processing of paired-end fastq files were performed according to the workflow in the previously reported TAS-seq method (Shichino S, Ueha S, Hashimoto S, Ogawa T, Aoki H, Wu B, et al. TAS-Seq is a robust and sensitive amplification method for bead-based scRNA-seq. Communications biology. 2022;5(1):602.). In brief, sorted cells were used to synthesize cDNA with BD Rhapsody Express Single-Cell Analysis System (BD Biosciences). cDNAs were amplified by the TAS-seq method at Immunogeneteqs Inc. (Noda city, Chiba, Japan) and sequenced with an Illumina Novaseq 6000 sequencer (Illumina, San Diego, CA, USA), using a Novaseq 6000 S4 reagent kit v1.0 or v1.5.