title: GSM8965999: Rhesus monkey-ID AM365 - ACC; Macaca mulatta; RNA-Seq
library_construction_protocol: ACC and LPFC tissue samples that were previously flash-frozen and stored at -80oC were dissociated by Dounce homogenization in lysis buffer (250mM sucrose, 25mM KCl, 5mM MgCl2, 10mM Tris buffer, pH 8.0, 0.1% Triton X-100, 1µM DTT, 1x Protease inhibitor, 1x RNase inhibitor and 1µM DAPI). The homogenate was then filtered through a 40µm-cell strainer to collect single nuclei. Fluorescence-activated cell-sorting (FACS) was performed at the Chobanian and Avedisian BUSOM Flow Cytometry Core Facility on the BD FACS ARIA II SORP (Becton Dickson) to remove cellular debris and to capture singlets by gating on forward and side-scatter light properties and nuclei fluorescent expression of DAPI. We collected 50,000 nuclei into a collection buffer (0.04% Bovine Serum Albumin (BSA), in PBS supplemented with 1x Protease inhibitor, and 1x RNase inhibitor). The sorted nuclei were then processed at the Chobanian and Avedisian BUSOM Microarray and Sequencing Resource Core Facility using the Chromium droplet-based platform (10X Genomics) whereby single nuclei, reagents, and a single gel bead containing barcoded oligonucleotides are encapsulated into nanoliter-sized Gel Bead-in-Emulsion (GEMs) using the GemCode platform for downstream reverse transcription of RNAs. Full length, barcoded cDNA was then amplified by polymerase chain reaction (PCR) for the generation of snRNA-seq libraries. The resulting cDNA libraries were assessed via Bioanalyzer High Sensitivity DNA Assay (Agilent Technologies, USA) and sequences on an Illumina NextSeq500 instrument with the target capture of 2000 cells per sample in accordance with the Illumina and 10x Genomics guidelines with 1.4-18.8 input and 1% PhiX control library spike-in (Illumina, USA).