GSM5009621: P3; Mus musculus; ATAC-seq
The testes were collected and with tunica albuginea removed. The testes were then digested into single cell suspension with type 4 collagenase and hyaluronidase. Cell tagmentation was performed according to SureCell ATAC-Seq Library Prep Kit (17004620, Bio-Rad) User Guide (10000106678, Bio-Rad) and the protocol based on Omni-ATAC was followed (Corces et al. 2017). In brief, washed and pelleted cells were lysed with the Omni-ATAC lysis buffer containing 0.1% NP-40, 0.1% Tween-20, 0.01% digitonin, 10 mM NaCl, 3 mM MgCl2 and 10 mM Tris-HCl pH 7.4 for 3 min on ice. The lysis buffer was diluted with ATAC-Tween buffer that contains 0.1% Tween-20 as a detergent. Nuclei were counted and examined under microscope to ensure successful isolation. Same number of nuclei were subjected to tagmentation with equal ratio of cells/Tn5 transposase to minimize potential batch effect. Nuclei were resuspended in tagmentation mix, buffered with 1×PBS supplemented with 0.1% BSA and agitated on a ThermoMixer for 30 min at 37 °C. Tagmented nuclei were kept on ice before encapsulation. Tagmented nuclei were loaded onto a ddSEQ Single-Cell Isolator (Bio-Rad). scATAC-Seq libraries were prepared using the SureCell ATAC-Seq Library Prep Kit (17004620, Bio-Rad) and SureCell ATAC-Seq Index Kit (12009360, Bio-Rad). Bead barcoding and sample indexing were performed with PCR amplification as follows: 37 °C for 30 min, 85 °C for 10 min, 72 °C for 5 min, 98 °C for 30 s, eight cycles of 98 °C for 10 s, 55 °C for 30 s and 72 °C for 60 s, and a single 72 °C extension for 5 min to finish. Emulsions were broken and products were cleaned up using Ampure XP beads. Barcoded amplicons were further amplified for 8 cycles. PCR products were purified using Ampure XP beads and quantified on an Agilent Bioanalyzer (G2939BA, Agilent) using the High-Sensitivity DNA kit (5067-4626, Agilent). Libraries were sequenced on HiSeq 2000 with 150 bp paired-end reads.