title: NextSeq 550 paired end sequencing; snRNA-seq of gorilla (Gorilla gorilla) adult testis
design: snRNA-seq of gorilla (Gorilla gorilla) adult testis
library_construction_protocol: Tissue pieces were homogenized in prechilled 250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM HEPES pH 8, 1 %, BSA, 0.1 % IGEPAL, 1 µM DTT, 0.4 U/µL RNaseIn, and 0.2 U/µL Superasin and lysed for 5 min on ice. The lysate was centrifuged at 100 x g for 1 min at 4 °C. The supernatant was transferred to a new reaction tube and centrifuged at 500 x g for 5 min at 4 °C. The supernatant was removed and the pellet resuspended in 1 mg/mL DSP in PBS and incubated for 30 min at room temperature. The fixation was quenched by addition of Tris-HCl to a final concentration of 20 mM. The fixed suspension was pelleted at 500 x g for 5 min at 4 °C. The supernatant was removed and the pellet resuspended in 250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM TRIS-HCl pH 8, 1 %, BSA, 1 µM DTT, 0.4 U/µL RNaseIn, and 0.2 U/µL Superasin. This was centrifuged at 500 x g for 5 min at 4 °C. The supernatant was removed and the pellet resuspended in PBS. Nuclei were isolated through a combination of mechanical and chemical lysis. Per library 15,000 – 20,000 nuclei were loaded into one channel of the ChromiumTM Single Cell A Chip (10X Genomics®). Libraries were prepared using Chromium Single Cell 3′ Library & Gel Bead Kit v2 according to manufacturer's instructions.