Bulk ATAC-Sequencing of isolated spermatocytes from testis tissue from B6/CAST F1 hybrid mice
Bulk ATAC-Sequencing of isolated spermatocytes from testis tissue from B6/CAST F1 hybrid mice
Cell Isolation for ATAC-Seq of murine testicular tissue using the 10x Genomics platform was performed similarly to Ernst et al, 2019. Tissue digestion was performed using 5mg/ml Collagenase A, 5 mg/ml Dispase II and 2.5 mg/ml DNAse I. After preparation of single cell suspensions from testes, cells were stained with 5mug/mul Hoechst 33342 (R37165, ThermoFisher) for 45 minutes at 37°C. Cells were resuspended in phosphate buffered saline (PBS, Sigma) with 1% Fetal Calf Serum (FCS, Gibco, 16140071) with propidium iodide (P4170) at a final concentration of 1 μg/ml, and 50.000 cells were sorted on a BD FACSAria Fusion machine. We performed bulk ATAC-Seq as described with modifications (Corces et al. 2017). Cells were washed with 500μl ice-cold PBS and incubated for 3 minutes in 50μl cell lysis buffer (10mM Tris-HCl pH 7.5 (AM9850G, LIFE Technologies), 10mM NaCl (AM9760G, ThermoFisher), 3mM MgCl (AM9530G, ThermoFisher), 0.1% NP-40 (85124, LIFE Technologies), 0.1% Tween-20 (P1379, Sigma), 0.01% Digitonin (BN2006, LIFE Technologies) ) on ice. 1ml wash buffer (10mM Tris-HCl pH 7.5, 10mM NaCl, 3mM MgCl, 0.1% Tween-20) was added, cells were centrifuged for 10 minutes at 500g and 4°C. Transposition of nuclei was performed by adding 50mul transposition mix (25mul 2x TD buffer (Illumina, 20034197), 16.5mul PBS, 0.5mul 10% Tween-20, 0.5mul 1% Digitonin, 2.5mul tagment DNA enzyme (Illumina, 20034197)) to the pellet and incubation at 37°C for 30 minutes. After incubation, DNA was isolated using MinElute PCR Purification Kit (28006, Qiagen) and libraries were PCR-amplified using NEBNext® High-Fidelity 2X PCR Master Mix (M0541S, NEB) with appropriate primers.