Magnetic cell sorting separator

Interstitial cell dissociations were performed by modifying digestion buffer 1 – which replaces 2.5mg of collagenase 1a1 with 0.25mg of collagenase D (Sigma). This milder digest is sufficient to gently dissociate the interstitial cells, but the second digestion buffer containing trypsin is the same for all sample preps since this is required to dissociate the seminiferous tubule into a single cell suspension. For Thy1+ enrichment, we performed a magnetic cell-sorting separator (Miltenyi Biotec) using anti-CD90.2 (Miltenyi Biotec) antibody. Cells were additionally stained with anti-CD90-FITC conjugated (1:200; Abcam, Cat# ab62009; RRID: AB_940927) or anti-CD90-PerCP cyanine5.5 (1:200; Life Technologies, Cat# 45090082; RRID: AB_2573662) for 20 min prior to flow cytometry and CD90+ cells were collected for Drop-seq. For Sca1+ (Ly6a) cell enrichment, cells were stained with anti-Ly6a-PerCP cyanine5.5 (1:200; Life Technologies, Cat# 45-5981-82; RRID: AB_914372) for 20 min or biotinylated anti-Ly6a (1:200) for 20 min followed by streptavidin conjugated Alexa Fluor 488 (1:1000; Life Technologies Cat# S11223; RRID: AB_2336881) for 20 min prior to flow cytometry. Sca1+ cells were collected for Drop-seq.

Spermatogonia were enriched by selection of C-kit+ or Gfra1+ cells. C-kit+ (CD117) cells were isolated from whole tubule cell suspensions on a magnetic cell-sorting separator (Miltenyi Biotec) using an anti-CD117 (Miltenyl Biotec) antibody. Cells were additionally stained with a biotinylated anti-CD117 antibody (1:200) for 20 min followed by streptavidin conjugated Alexa Fluor 488 (1:1000, Life Technologies) for 20 min prior to flow cytometry. For Gfra1+ selection, tamoxifen inducible Gfrα1^CreERT2 mice on a C57BL/6 background (kindly provided by Dr. Shosei Yoshida, National Institute for Basic Biology, Okazaki, Japan) were crossed with B6.Gt(ROSA)26Sor^{tm4(ACTB-tdTomato,-EGFP)Luo} (Rosa^mT/mG; JAX®mice, stock no. 007676). Labeling of Gfra1+ spermatogonia was conducted by injecting a Gfrα1^CreERT2; Rosa^mT/mG mouse with 2mg of 4OH-tamoxifen (dissolved in ethanol and then in corn oil, Sigma) per day for 3 weeks prior to euthanasia at 8 weeks of age. All live Gfra1 positive (GFP expressing) and tdTomato negative cells were collected by flow cytometry. The extended labeling was necessary to obtain the number of cells needed for Drop-seq. As a result this dataset includes spermatogonia and spermatocytes.