Freshly collected testicular cells at E14.5 from 12 controls and 16 Nr5a1 Sertoli cell deficient mutants. The testes were incubated for 10 minutes at 37°C in 350 μl of trypsin/EDTA 0.05% (w/v), phenol red solution (Gibco Ref 25300), filtered through a 40 μm cell strainer to generate single cell suspensions, centrifuged at 3000g and suspended in 300 µl PBS. Cell number and viability were determined by a Trypan Blue exclusion assay on a Bürker Chamber. Samples consisted of > 90 percent viable cells were processed on the Chromium Controller from 10X Genomics (Leiden, The Netherlands). 10000 total cells were loaded per well to yield approximately 6000 captured cells into nanoliter-scale Gel Beads-in-Emulsion (GEMs). Single cell 3 prime mRNA seq library were generated using Chromium Single Cell 3 prime Reagent Kits v3 (10X Genomics). Briefly, GEMs were generated by combining barcoded gel beads, a RT master mix containing cells, and partitioning oil onto Chromium Chip B. Following full length cDNA synthesis and barcoding from poly-adenylated RNAs, GEM were broken and pooled before cDNA amplification by PCR using 11 cycles. After enzymatic fragmentation and size selection, sequencing library were constructed by adding P5 and P7 primers (Illumina, San Diego, USA) as well as sample index via end-repair, A-tailing, adaptor ligation and PCR amplification with 10 cycles. Library quantification and quality control was performed using Bioanalyzer 2100 (Agilent Technologies, Santa Clara CA).