title: GSM9115487: DMRT1_CUTTag_P8_Aundiff_Smarca5_ctrl_rep2; Mus musculus; OTHER
library_name: GSM9115487
library_construction_protocol: 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. CUT&Tag libraries of ctrl Aundiff and ProSG for SMARCA5 were prepared as previously described (a step-by-step protocol https://www.protocols.io/view/bench-top-cut-amp-tag-kqdg34qdpl25/v3) using CUTANA™ pAG-Tn5 (Epicypher, 15-1017). Quantitative spike-in CUT&Tag for DMRT1 of Smarca5-ctrl and cKO Aundiff was performed by adding Drosophila S2 cells at a 1:5 ratio to mouse spermatogonial cells (5,000 S2 cells to 25,000 mouse cells) in each reaction. The antibodies used were rabbit anti-SMARCA5 (1/100) or rabbit anti-DMRT1 antibody (1/50). CUT&Tag libraries were sequenced on the Novaseq X Plus system with 150-bp paired-end reads.
identifiers.namespace: GEO
identifiers.id: GSM9115487_r1