GSM6856439
Testicular cells were prepared individually from three different adult mice (WT, KO and KI) following two-step enzymatic digestion. Testes from each mouse were dissected and tunica albuginea was removed. The seminiferous tubules were treated with collagenase type-I in M199 medium containing 0.1% bovine serum albumin (BSA) for 10 mins at 35°C and dispersed by gently shaking in between. The collagenase treated tubules were minced and incubated in M199 with 0.1% BSA and 0.1% trypsin for 10 mins at 35°C in rotation at 100 rpm to obtain dispersed cell suspension. The trypsin digestion was stopped by adding 0.02% of trypsin inhibitor (Sigma) to the sample. Cells were filtered through 100 µm and then with 40 µm cell strainer (Millipore) to obtain single cell suspension fraction, pelleted at 800g for 2 mins, washed and resuspended in cold M199 with 0.1% BSA for scRNA-seq. scRNA-seq libaray of testicular cells was prepared according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). In brief, freshly prepared cell suspension (7,000 cell for each sample) was loaded into Chromium microfluidic chips used to generate single-cell gel bead emulsions using the 10X Genomics Chromium controller as per manufacturer's protocol. Each scRNA-seq library was constructed following the manufacturer's instructions. Sequencing of libraries was performed for 100 cycle paired-end run on an Illumina HiSeq 2500 instrument.