Protocols: 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 32 °C. Tubules were disrupted by gently pipetting up and down, followed by filtration through a 100µm 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 β-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.
ENA first public
2022-06-15
ENA last update
2022-06-15
External Id
SAMEA11937504
INSDC center alias
Laboratory of Functional Genomics and Bioinformatics Institute of Molecular Genetics of the ASCR, v. v. i.
INSDC center name
Laboratory of Functional Genomics and Bioinformatics Institute of Molecular Genetics of the ASCR, v. v. i.
INSDC first public
2022-06-15T12:17:12Z
INSDC last update
2022-06-15T12:17:12Z
INSDC status
public
Submitter Id
E-MTAB-11271:M1581
age
8
broker name
ArrayExpress
cell type
Sertoli cell
common name
house mouse
developmental stage
adult
genotype
wild type genotype
individual
3
organism part
testis
sample name
E-MTAB-11271:M1581
sex
male
strain
C57BL/6