GSM5412016: dlPFC single-nucleus transcriptomes from 65Z; Macaca mulatta; RNA-Seq
We generated nuclei suspensions from frozen tissue biopsies following methods described by Cao et al. 2019 (PMID: 30787437, series accession: GSE119945), with the following modifications. We first homogenized samples by thoroughly grinding samples with a pre-chilled disposable pestle (Fisher Scientific cat. no. 12-141-364) in 50 µl of cell lysis buffer on ice. We brought the volume to 1 ml with ice-cold cell lysis buffer and filtered the mixture using a 70 µm cell strainer (pluriSelect cat. no. 43-10070-70). The nuclei were then fixed in 4% paraformaldehyde (6 ml reaction volume) for 15 minutes with gentle mixing, washed once in 1 ml of ice-cold nuclei wash buffer, then resuspended in 200 µl of nuclei wash buffer. After counting a small aliquot with a 1:1 trypan blue stain using the Countess II system, nuclei were allocated in 1-million-nucleus aliquots and flash frozen in liquid nitrogen. Methods for sci-RNA-seq3 library preparation and sequencing followed those described by Cao et al. (PMID: 30787437, series accession: GSE119945). Briefly, nuclei were thawed in a 37°C water bath for 5 minutes, spun down at 500 × g for 5 minutes, and incubated in 400 µl of permeabilization buffer (nuclei wash buffer supplemented with 0.2% Triton X-100) for 3 minutes. Nuclei were washed, sonicated for 12 sec on low power mode (Diagenode), washed, and resuspended in 100 µl of nuclei wash buffer. The sci-RNA-seq3 prep follows the Cao et al. methods for paraformaldehyde-fixed nuclei with the following modifications: samples were counted with the ImageExpress Pico System (Molecular Devices), 2 µl of oligo-dT primers were added to each well with 80,000 nuclei for reverse transcription, Quick Ligation kit (New England Biolabs) was used in place of T4 ligase, and tagmentation was performed using 1/40th µl per well of i7 loaded TDE1 enzyme, prepared at the University of Washington following Hennig et al. 2018 (PMID: PMID: 29118030).