GSM9115495: ATACseq_P8_Adiff_Smarca5_ctrl_rep2; Mus musculus; ATAC-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. ATAC-seq libraries of germ cells were prepared as described; briefly, 10,000 Aundiff, Adiff or P0 prospermatogonia (ProSG) were pooled from two independent mice as one replicate. Samples were lysed in 50 μl of lysis buffer (10 mM Tris–HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2 and 0.1% NP-40, 0.1% Tween-20, and 0.01% Digitonin) on ice for 5 min. Immediately after lysis, the samples were spun at 500 × g for 10 min at 4°C and the supernatant was removed. The sedimented nuclei were then incubated in 10 μl of transposition mix (0.5 μl homemade Tn5 transposase (∼1 μg/μl), 5 μl 2× tagment DNA buffer (10 mM Tris–HCl (pH 7.6), 10 mM MgCl2 and 20% dimethyl formamide), 3.3 μl PBS, 0.1 μl 1% digitonin, 0.1 μl 10% Tween-20 and 1 μl water) at 37°C for 30 min in a thermomixer with shaking at 500 rpm. After tagmentation, the transposed DNA was purified with a MinElute kit (Qiagen). Polymerase chain reaction (PCR) was performed to amplify the library using the following conditions: 72°C for 3 min; 98°C for 30 s; thermocycling at 98°C for 10 s, 60°C for 30 s and 72°C for 1 min. Quantitative PCR was used to estimate the number of additional cycles needed to generate products at 25% saturation. Seven to eight additional PCR cycles were added to the initial set of five cycles. Amplified DNA was purified by 1.0x SPRIselect beads (Beckman Coulter). ATAC-seq libraries were sequenced on the HiSeq X Ten system with 150-bp paired-end reads.