title: GSM9235488: H3K27ac_CUTRun_4wk_PS_Daxx_cKO_rep1; Mus musculus; OTHER
library_name: GSM9235488
library_construction_protocol: Isolation of pachytene spermatocytes and round spermatids using fluorescence-activated cell sorting (FACS) was performed using a SONY SH800S instrument, with Vybrant DyeCycle Violet Stain (DCV) (Invitrogen, V35003) stained testicular single-cell suspensions prepared as described previously (Yeh et al. 2021).  In brief, to prepare single cells suspensions for cell sorting, detangled seminiferous tubules from adult mouse testes were first washed with 1× Hanks' Balanced Salt Solution (HBSS, Gibco, 14175095) 3 times and then incubated with 1 mg/mL collagenage type 1 (Worthington, CLS1) for 20 min at 35 °C to remove interstitial cells. Then, the tubules were digested in Dulbecco's Modified Eagle Medium (DMEM, Gibco, 11885076) containing 2% Fetal Bovine Serum (FBS, Gibco, 16000044), 2 mg/mL collagenase type 1, 0.25 mg/mL DNase I (Sigma, D5025), 1.5 mg/mL hyaluronidase (Sigma, H3506) and 700 U/ml recombinant collagenase (FUJIFILM Wako Pure Chemical Corporation, 036-23141) at 35 ℃ for 20–30 min with vigorous pipetting for cell dissociation. The floating germ cell-enriched cell suspension was then collected from plates and washed with 10 mL FACS buffer (PBS containing 2% FBS) 3 times by centrifugation at 300 × g for 5 min and filtered through a 70 µm nylon cell strainer (Falcon, 352350). The resultant single cells were stained with DCV. After 30 min incubation at 35 ℃, cells were filtered into a 5 mL FACS tube through a 35 µm nylon mesh cap (Falcon, 352235). Samples were kept on ice until sorting. Daxx-ctrl or Daxx-cKO sorted 50,000 PS or 200,000 RS from one individual mouse as one replicate, and two independent biological replicates were used for CUT&RUN library generation as previously described (a step-by-step https://www.protocols.io/view/cut-amp-run-targeted-in-situ-genome-wide-profiling-14egnr4ql5dy/v3). The antibodies used were mouse anti-Histone H3.1/3.2 (1/100; Active Motif AM61629), mouse anti-Histone H3.3 (1/100; Active Motif AM91191), rabbit anti-H3K9me3 (1/100; Abcam ab8898), rabbit anti-H3K27ac antibody (1/100; Cell Signaling Technology; 8173), and mouse anti-Histone H3t (1/100; requested from Dr. Yasuyuki Ohkawa, generated from Ueda et al. 2017). Libraries were constructed using NEBNext® Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S). Prepared CUT&RUN libraries were sequenced on the NovaSeq X Plus system (Illumina) with paired-ended 150-bp reads.