Integrated single-cell chromatin profiling of testicular cell types from the cynomolgus macaque reveals functional gene regulatory networks during spermatogenesis
Integrated single-cell chromatin profiling of testicular cell types from the cynomolgus macaque reveals functional gene regulatory networks during spermatogenesis
Macaca fascicularis testis tissue was 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 40uM strainer in order to obtain a suspension of single-cells. Dissociated unsorted testes cells were cryopreserved in DMEM with 20% FBS and 10% DMSO, at a concentration of 5000 cells/uL. Suspended cells were let to stand on ice for 15 minutes before moving to a chilled cryo-freezing container. The container was then placed in a -80 °C freezer. Cells were stored in -80 °C freezer for long term storage.  Cells were thawed in the water bath at 37 °C for 1min. Cells were then transferred to a 50mL tube. Cryovials were rinsed with 1mL of prewarmed DMEM with 10% FBS slowly added to the tube. Cells were diluted sequentially by incremental 1:1 volume additions of cell media 5 times with 1min wait intervals between additions. Tubes were then centrifuged at 300rcf for 5min. For 10X Genomics Single Cell ATAC-Seq, supernatant was removed, and cell pellets were resuspended in 1mL of PBS with 0.04% BSA and transferred to 2mL tubes. Tubes were centrifuged again at 300rcf for 5min. The supernatant was removed, and pellets were resuspended in 500uL of PBS with 0.04% BSA. Cell suspensions were then passed through a 40um cell strainer, and cell concentration was determined using a haemacytometer.  With an estimate nuclei recovery of 6000, 2.2uL nuclei suspension and 2.8uL of Diluted Nuclei Buffer was added to 10uL of Transposition Mix.  The mixture was incubated in a thermal cycler at 37 °C for 1h and held at 4 °C. Transposed nuclei were subsequently loaded into each channel of a Chromium Next GEM Chip H (10x Genomics, PN-1000162). A Chromium Next GEM Single Cell ATAC Library & Gel Bead Kit v1.1 (10x Genomics, PN-1000176) was used for GEM generation, barcoding and library construction, following manufacturer's protocol (Chromium Next GEM Single Cell ATAC Reagent Kits User Guide, v1.1 Chemistry).