title: GSM8114648: Whole testis, synchronized, 763.2hr, EP4; Mus musculus; RNA-Seq
library_construction_protocol: To create a single cell suspension, testes were detunicated and placed in 10 mL of collagenase solution (1mg/mL, X7H9763A, Worthington Biochemical, Lakewood, NJ, USA) and 1mL DNase solution (7mg/mL, 9003-98-9, Sigma, St. Louis, MO, USA) diluted in 1x HBSS (Thermo Fisher, Waltham, MA, USA) in a 50mL conical. Tubules were dissociated by pipetting up and down once with a 10mL serological pipet, then swirling the tube several times before incubating the tubules in a 37°C water bath for three minutes. After incubation, testes were swirled again vigorously and incubated for three additional three-minute periods. Tubules were then placed on ice for 1 minute to allow the tubules to settle to the bottom of the conical, and the collagenase/DNase solution was removed. Tubules were then rinsed with 10mL 1x HBSS, swirled, again set on ice for one minute to allow the tubules to settle, and the supernatant was removed. This process was repeated three additional times and then a 9mL 0.25% trypsin and 1mL DNase solution was added and the resulting mixture was then pipetted up and down 10 times with a p1000 micropipette to dissociate the tubules further before being placed in a 37°C water bath for five minutes. This step was repeated, with another 1mL of the DNase solution being added when necessary to support tubule degeneration, another five times. 10% of the total volume of fetal bovine serum (FBS) was then added and cells were put through at 40μM cell strainer that was prerinsed with 1mL 1x HBSS. The 50mL conical that had previously contained the cells was then rinsed three times with 1x HBSS to collect any lingering cells, and this solution was also sent through the 40μM cell strainer. Cell counts were estimated using a hemocytometer, spun down at 600 × g for 7 min at 4°C, and resuspended at 4 million cells/mL in 10% DNase in 1x DPBS (ThermoFisher, Waltham, MA, USA). To determine cell viability, 20µL of the cell suspension was diluted 1:1 with 0.4% trypan blue (Invitrogen, Waltham, MA, USA), and on average the cell suspensions used for scRNA-seq library had a live cell percentage of at least 96.95% (+/- 1.13%). Cells from triplicate males were pooled at equal proportions and loaded into a Chromium Controller (10× Genomics, Inc., Pleasanton, CA, USA). Single-cell cDNA libraries were generated using v3.1 chemistry according to the manufacturers protocol (10× Genomics, Inc.) and sequenced via NovaSeq PE150 (Novagene, Sacramento, California). Raw data were processed, demultiplexed, and aligned to the 10-mm mouse transcriptome using the 10× Genomics Cell Ranger pipeline (v3.0.2)