ATAC-seq of mouse spermatogonia cells at two developmental stages (PND15 and adult)
ATAC-seq of mouse spermatogonia cells at two developmental stages (PND15 and adult)
For pup testis, dissociated cells were stained with BV421-conjugated anti-β2M, biotin-conjugated anti-THY1 (53-2.1), and PE-conjugated anti-αv-integrin (RMV-7) antibodies. THY1 was detected by staining with Alexa Fluor 488-Sav. For adult testes, cells were stained with anti-α6-integrin (CD49f; GoH3), BV421-conjugated anti-β2 microglobulin (β2M; S19.8), and R-phycoerythrin (PE)-conjugated anti-THY1 (CD90.2; 30H-12) antibodies. α6-Integrin was detected by Alexa Fluor 488-SAv after staining with biotin-conjugated rat anti-mouse IgG1/2a (G28-5) antibody. Prior to FACS, 1 μg/ml propidium iodide (Sigma) was added to the cell suspensions to discriminate dead cells. All antibody incubations were performed in PBS-S for at least 30 min at 4oC followed by washing in PBS-S. Antibodies were obtained from BD Biosciences (San Jose, United States) unless otherwise stated. Cell sorting was performed at 4oC on a FACS Aria III 5L using an 85μm nozzle at the Cytometry Facility of University of Zurich. For OmniATAC on PND15 spermatogonia, 25'000 cells were collected in a separate tube, pelleted by centrifugation and immediately processed using the OmniATAC library preparation protocol (Corces et al., 2017). For OmniATAC on adult spermatogonia, 5000 cells from each animal were collected in a separate tube and further processed using the same protocol.  Chromatin accessibility was profiled from PND15 and adult spermatogonial cells. The libraries were prepared according to the Omni-ATAC protocol, starting from 25 000 PND15 and 5000 adult sorted spermatogonia, respectively (Corces et al., 2017). Briefly, sorted cells were lysed in cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin) and the nuclei were pelleted and transposed using the Nextera Tn5 (Illumina) for 30 min at 37oC in a thermomixer with shaking at 1000 rpm. The transposed fragments were purified using the MinElute Reaction Cleanup Kit (Qiagen).  Following purification, libraries were generated by PCR amplification using the NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs), and purified using Agencourt AMPure XP magnetic beads (Beckman Coulter) in order to remove primer dimers (78bp) and large fragments of 1000-10,000 bp in length. Library quality was assessed on an Agilent High Sensitivity DNA chip using the Bioanalyzer 2100 (Agilent Technologies). 6 samples were sequenced from PND15 and 5 from adult spermatogonial cells.