GSM6592479: A3-2; Mus musculus; RNA-Seq
Single Cell Suspension  Testes were removed and placed in Krebs-Ringer Bicarbonate Buffer (EKRB). For each experiment, testes from at least 3 adult males or 5 pups from a given litter were detunicated and transferred to a solution containing 4.5 ml of 0.25% trypsin (Gibco) and 0.5 ml DNase1 (7 mg/ml, Sigma-Aldrich) and incubated in a 37°C water bath for 3 min. Then, additional 1 ml of DNase1 was added and incubated in a 37°C water bath for another 3 min. An additional 1 ml DNase1 was added and the mixture was pipetted up and down to further break up the testes, followed by a 1 min incubation in a 37°C water bath. One ml of fetal bovine serum (FBS, ATCC) was added to deactivate the trypsin, and the mixture was triturated to break apart any remaining clumps of intact testis. The cell mixture was filtered through a 40 μm sieve and centrifuged at 600 x g for 7 min. The resulting cell pellet was suspended in either sorting media containing 3% FBS + 10mM EDTA + 10mM HEPES in PBS and used for FACS or fixed in ethanol for ploidy analysis.     Fluorescence-activated cell sorting (FACS)  EGFP+ germ cells from Uchl1-eGfp mice were sorted on a Becton Dickinson AriaFusion cell sorter. A 100 mW 488 nm laser was used for excitation of the EGFP signal, and a 530/30 bandpass filter was used for detection of the emitted fluorescence. Dead cells were removed using forward and side scatter gating as well as fluorescence gating on cells excluding DAPI (Thermo Fisher #D1306). Single DNA-containing cells were identified using DRAQ5 fluorescent probe (Thermo Fisher #62254). Doublets were removed using FSC-height vs FSC-area plots. An 85 m nozzle was used for sorting, and the flow rate was controlled to 5,000-9,000 events/sec. Cells were recovered in sorting buffer (15% FBS + 10 mM EDTA + 10 mM HEPES in 1X PBS) in 5 ml polypropylene tubes (Corning LS, 352063) pre-coated with 10% BSA. Cell purity was assessed by reanalyzing a small aliquot of the sorted cells by immunostaining.     RNA Preparation:  Males with synchronized spermatogenesis were euthanized from mice at P17 for isolation of type B spermatogonia and at P19 for isolation of preleptotene spermatocytes. The latter were obtained from mice with an RA-sufficient and -deficient environment. FACS was used to isolate 2-3 x 106 type B spermatogonia and 2-3 x 106 preleptotene spermatocytes (3-4 males, each litter). Total RNA was isolated from cells using the GeneJet RNA purification kit (Thermo Scientific). Libraries were produced with the Illumina TruSeq Stranded mRNA kit according to manufacturer recommendations.