NextSeq 500 sequencing; RNA-seq to investigate the FBXO38 knockout in mouse Sertoli cells
RNA-seq to investigate the FBXO38 knockout in mouse Sertoli cells
Dissected testes were decapsulated and processed into a single-cell suspension using a previously described protocol (Bellve et al., 1977). Briefly, seminiferous tubules were submerged in EKRB medium (120.1mM NaCl, 4.8mM KCl, 25.2mM NaHCO3, 1.2mM KH2PO2, 1.2mM MgSO4, 1.3mM CaCl2, 11mM glucose, 1mM L-glutamine, 4.4mM sodium lactate, 6.5mM sodium pyruvate, 20mM HEPES, non-essential amino acid and penicillin-streptomycin) with collagenase (0.5 mg/ml) and incubated for 20 minutes at 32C. Tubules were disrupted by gently pipetting up and down, followed by filtration through a 100m strainer. Cells were sedimented at 300g for 10 minutes and resuspended in fresh EKRB medium. The cell suspension was stained with a commercially available antibody against the follicle-stimulating hormone receptor (FSHR) conjugated with phycoerythrin (Bioss, BS-20658R-PE) for 1 hour. FSHR-positive Sertoli cells were sorted (Influx; BD Biosciences) into the RLT buffer (Qiagen) supplemented with -mercaptoethanol. Fbxo38 knockout mice were generated in a C57BL/6N background by electroporating the Cas9 protein and specific sgRNAs targeting Fbxo38 into the zygotes. Selected founder was backcrossed for two generations to C57BL/6N mice to obtain homozygous animals. RNA was extracted using RNeasy Micro Kit (Qiagen). Libraries were prepared with Takara SMARTer Stranded Total RNA-Seq Kit v2.