GSM9115502: RNAseq_P8_Adiff_Smarca5_ctrl_rep1; Mus musculus; RNA-Seq
To prepare single cell suspensions for cell sorting, detangled seminiferous tubules were incubated in 1× Krebs–Ringer Bicarbonate Buffer (Sigma, K4002) supplemented with 1.5 mg/ml collagenase Type 1 and 0.04 mg/ml DNase I at 37°C for 15 min with gentle agitation and dissociated using vigorous pipetting, and then add 0.75 mg/ml hyaluronidase (Sigma, H3506) and incubated at 37°C for 10 min with gentle agitation and dissociated using vigorous pipetting. The cell suspension was centrifuged at 300 × g, the cells resuspended in 10 ml FACS buffer (PBS containing 2% FBS) and then centrifuged again at 300 × g for 5 min, and this step was repeated one more time. The pelleted cells were resuspended 1 ml FACS buffer and then filtered through a 70 μm nylon cell strainer (Falcon, 352350). The resultant single cells were stained with cocktails of antibodies diluted with FACS buffer listed as follows: PE-conjugated anti-mouse/human CD324 (E-cadherin) antibody (1:500, Biolegend, 147303), PE/Cy7-conjugated anti-mouse CD117 (c-Kit) antibody (1:200, Biolegend, 105814) and FITC-conjugated anti-mouse CD9 antibody (1:500, Biolegend, 124808). After a 50 min incubation on ice, cells were washed with 10 ml FACS buffer three times by centrifugation at 300 × g for 5 min and filtered into a 1 ml FACS tube through a 35 μm nylon mesh cap (Falcon, 352235). 7-AAD Viability Stain (Invitrogen, 00-6993-50) was added to cell suspension for the exclusion of dead cells. Samples were kept on ice until sorting. Cell sorting was performed using SH800S (SONY). Cells were analyzed after removing small and large debris in FSC-A versus SSC-A gating, doublets in FSC-W versus FSC-H gating, and 7AAD+ dead cells. Then the desired cell population was collected in gates determined based on antibody staining. 10,000 Aundiff or Adiff cells were pooled from two independent mice as one replicate, and two independent biological replicates were used for RNA-seq library generation. Total RNA was extracted using the RNeasy Plus Micro Kit (QIAGEN, Cat # 74034) according to the manufacturer's instructions. Library preparation was performed with NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® (NEB, E6420S) according to the manufacturer's instructions. Prepared RNA-seq libraries were sequenced on the HiSeq X Ten system (Illumina) with paired-ended 150-bp reads.