title: Illumina NovaSeq 6000 sequencing; Single-cell RNA-Sequencing of testis tissue from 3 mouse species and their immediate crosses
design: Single-cell RNA-Sequencing of testis tissue from 3 mouse species and their immediate crosses
library_construction_protocol: All mice used in this study were derived from colonies maintained inhouse, at around 8 weeks of age after spermatogenesis is fully established. For the cross-species analysis, we used animals from the C57BL/6, CAST/EiJ and CAROLI/EiJ strains. For the F1 analysis, we used C57BL/6-Ly5.1 and CAST/EiJ strains as well as F1 mice derived from a C57BL/6-Ly5.1 (female) x CAST/EiJ (male) cross. scRNA-Seq of murine testicular tissue using the 10x Genomics platform was performed similarly to Ernst et al, 2019. For the cross-species dataset, single-cell suspensions were generated by enzymatic digestion using 25 mg/ml Collagenase A (Sigma, 10103578001), 25 mg/ml Dispase II (Sigma, D4693) and 2.5 mg/ml DNAse I (Sigma, 10104159001) I for 30 min at 37C. For the F1 dataset, digestion was performed using 5mg/ml Collagenase A, 5 mg/ml Dispase II and 2.5 mg/ml DNAse I. After generation of single-cell suspensions, libraries were generated using 10x library construction kits according to the manufacturers instructions. For the cross-species dataset, for each sample, 10,000 cells were loaded into one channel of the Chromium™ Single Cell A Chip (10X Genomics ®, 1000009) and scRNA-Seq libraries were generated using the Chromium™ Single Cell 3' Library & Gel Bead Kit v2 (10X Genomics ®, 120237). For the F1 dataset, libraries were generated using the Chromium™ Single Cell B Chip (10X Genomics ® 1000073) and Single Cell 3' Library & Gel Bead Kit v3 (10X Genomics ®, 1000075). After generation of single-cell suspensions, libraries were generated using 10x library construction kits according to the manufacturers instructions. For the cross-species dataset, for each sample, 10,000 cells were loaded into one channel of the Chromium™ Single Cell A Chip (10X Genomics ®, 1000009) and scRNA-Seq libraries were generated using the Chromium™ Single Cell 3' Library & Gel Bead Kit v2 (10X Genomics ®, 120237). For the F1 dataset, libraries were generated using the Chromium™ Single Cell B Chip (10X Genomics ® 1000073) and Single Cell 3' Library & Gel Bead Kit v3 (10X Genomics ®, 1000075).