GSM6622128: control_testis_3; Mus musculus; RNA-Seq
Decapsulated testes were incubated in DMEM containing 0.75 mg/ml collagenase type IV (Gibco, 17104-019) for 10 mins at 37°C with occasional inversion. Equal volume of DMEM was added to the suspension of dissociated tubules before centrifuging at 400 xg for 5 mins at 4 °C. After removing the supernatant, the cell pellet was washed in DMEM and resuspended in 0.05% trypsin-EDTA (Gibco, 25200-056) for 10 mins incubation at room temperature. An equal volume of ice-cold DMEM+10%FBS was then added to the cell suspension before washing the pellet twice with media. The cell pellet was resuspended in DPBS (Sigma, D8537) containing 0.04% bovine serum albumin (sigma, A9647) and filtered through a 100 µm filter (Falcon, 22363549) then a 40 µm filter (Falcon, 352340) to generate a single cell suspension. Total RNA was extracted from dissociated testis cells by homogenizing in 1 ml of TRIzol reagent (Invitrogen, 15596026). After 5 mins of incubation, the lysate was mixed with 200µl of chloroform and centrifuged at 12,000 xg for 15 mins at 4°C. The aqueous phase was mixed with an equal volume of 100% ethanol and transferred to an RNeasy MinElute spin column (Qiagen, 74104) for purification according to the manufacturer's instructions. The quality of the eluted RNA was assessed by 28S/18S ribosomal ratios and RNA integrity number (RIN) using an Agilent Bioanalyzer. Libraries were constructed using the KAPA mRNA HyperPrep Kit (Roche # 08098123702) according to the manufacturer's instructions