GSM9066394: adultMaleThalamus, dominant, replicate_1; Mus musculus; RNA-Seq
Nuclear isolation, purification, and barcoding.  Nuclei were extracted following a custom protocol designed to minimize the amount of time from euthanasia to nuclear barcoding (approximately four hours). We prepared 10 biological samples for sequencing. Each biological sample was a pool of three mice (to obtain sufficient material for sequencing). Nine of the biological samples came from three hierarchy cohorts, each consisting of rank-1, rank-3, and control animals. The tenth biological sample was an additional control animal. Thus, the 10 biological samples were comprised of 30 animals. All animals were age-matched and processed in parallel. Mouse brains were quickly dissected on ice and 2mm thick coronal sections were cut on a brain matrix (Zivic Instruments, #5325). Sections were placed on a chilled surface under a dissecting microscope and tissue punches from the thalamus were collected using a 2mm diameter cylindrical corer (Fine Science Tools, #18035-02). Tissue punches were pooled in chilled Hibernate-A Medium (ThermoFisher, #A1247501), minced into approximately 0.52 mm pieces, and transferred to 3 mL lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.1% Nonidet P-40). Tissue was dounce homogenized in a glass tube 6-10 times with a grinder pestle (Potter-Elvehjem, #886000-0020) rotating at 300 rpm until opaque, transferred to a 15 mL tube, and pelleted in a swinging bucket centrifuge (Beckman Coulter GS-6R) at 50 RCF for 5 min at 4C. Supernatant was removed and tissue carefully resuspended in 1.5 mL PBS with BSA (1%) and RNase inhibitor (0.2 U/L, Promega, #N2615). To remove debris, the suspension was passed over a 70 M filter (ThermoFisher, #NC0120755) and then again over a 20 M filter (ThermoFisher, #130-101-812). With the suspension on ice, nuclei were labeled by adding DAPI (1.5 L, ThermoFisher, #D1306). DAPI-positive nuclei were sorted at 4C on a FACSAria flow sorter (BD Immunocytometry Systems) with a 70 mm nozzle on the four-way high speed purity setting until approximately 100,000 nuclei were collected. Using the 10X Genomics droplet microfluidics Gemcode platform 83, nuclei from the suspension were loaded onto Chromium instrument. Libraries were generated using Chromium v2 reagents, and cDNA amplified according to the manufacturer's instructions
GEO
GSM9066394_r1