Illumina HiSeq 4000 sequencing; Testis single-cell RNA-sequencing in the cynomolgus macaque reveals conserved transcriptional profiles during mammalian spermatogenesis
Testis single-cell RNA-sequencing in the cynomolgus macaque reveals conserved transcriptional profiles during mammalian spermatogenesis
Macaca fascicularis tetis tissues were incubated in dissociation buffer containing 200 U/mL collagenase I (Gibco, 17100017), for up to 5 mins with gentle agitation to dissociate tubules. The supernatant, containing mostly interstitial cells, was removed and fresh dissociation buffer containing collagenase, with 2.4 U/mL dispase II (Gibco, 17105041) and 1 mM CaCl2 in Hanks Balanced Salt Solution (HBSS) was added to the sample, and incubation was performed at 37 °C for 30 minutes in a water bath. Tubes were agitated with shaking every 5 minutes until tissue is dissociated fully into single cells. Enzymatic digest was quenched by the addition of 10 % FBS. Cells are then washed and resuspended in PBS, and filtered through a 40 μM strainer in order to obtain a suspension of single-cells. 4.8 ul of single-cell suspension at a concentration of ~1,000,000 cells/ml was loaded into one channel of the Chromium Single Cell Chip B (10X Genomics®), aiming for a recovery of 3000-4000 cells. Chromium Single Cell 3' Library & Gel Bead Kit using version 3 chemistry (10X Genomics, PN-1000092) was used for single cell barcoding, cDNA synthesis and library preparation, following manufacturer's protocol (Chromium Single Cell 3ʹ Reagent Kits v3 User Guide, Revision B).