GSM5456661: AR3; Mus musculus; ATAC-seq
Isolated hearts were washed with DPBS twice, and then collected in 1.5 mL EP tubes. Tissues were minced into pieces (~1 mm) on ice using scissors, and digested into single-cell suspensions with 1 mg/ml type II collagenase (17101015, GIBCO) and 1 mg/ml type IV collagenase (17104019, GIBCO) for 30min at 37 ℃ with intermittent shaking. The dissociated cells were separated and remaining undigested tissue were digested again with fresh digestion buffer. Digested suspension was passed through 70um strainer (Biologix).Dissociated cells were centrifuged at 300 g for 5 min at 4 °C, then re-suspended in 1 mL of cold DPBS with 0.1% BSA. After passing through a 40um cell strainer (Biologix), cells were washed twice, centrifuged at 300 g for 5 min at 4 °C.To isolate nuclei, 500,000 cell pellets were re-suspended in 100 uL chilled 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 - from (Corces, Trevino et al. 2017) supplemented with 1% BSA), and pipette mix 10X. After incubation for 5 min on ice, add 1 ml chilled Wash Buffer ((10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% BSA) to the lysed cells. Pipette mix 5x, then centrifuged at 300 g for 5 min at 4 °C. Based on number of cells used for isolation and assuming ~50% nuclei loss during cell lysis, resuspend in chilled Diluted Nuclei Buffer (PN-2000153, 10x Genomics). If cell debris and large clumps are observed, pass through a cell strainer. For low volume, use a 40 µm Flowmi Cell Strainer (H13680-0040, Bel-Art) to minimize volume loss. The scATAC library was prepared using the 10x Genomics platform with the Chromium Single Cell ATAC Library & Gel Bead Kit (10x Genomics, Pleasanton, California) as instructed by the manufacturer. A total of 15,000 nuclei per sample were used as input for single-cell ATAC-seq following the manufacturer's instructions. Briefly, after tagmentation, the cells were loaded on a Chromium Controller Single-Cell instrument to generate single-cell Gel Bead-In-Emulsions (GEMs) followed by linear PCR as described in the 10X scATAC-seq protocol using a Veriti 96-well thermal cycler (BioRad, 1851197). After breaking the GEMs, the barcoded tagmented DNA was purified with SPRIselect Reagent Kit (Beckman Coulter, Pasadena, CA) and further amplified to enable sample indexing and enrichment of scATAC-seq libraries. The final libraries were quantified using Bioanalyzer (Agilent) and QuBit (Thermofisher) analysis and then sequenced in MGISeq-2000FCL (MGI Tech Co., Ltd., China) with 100-bp paired-end read length targeting a depth of 30,000–50,000 reads per cell.
GEO
GSM5456661