GSM8636222: trnE-mutant, 3-week-old, 80% mutation load, mtscATAC-seq; Mus musculus; ATAC-seq
After obtaining the single-cell suspensions, we first fixed, lysed, and permeabilized the cells according to the method described in Nature Protocols (PMID:36792778). Briefly, the cells were fixed in 1% paraformaldehyde at room temperature for 10 minutes, with the tube being vortexed and inverted occasionally. The fixation was terminated with 0.125 M glycine. The cells were then lysed on ice for 3-5 minutes using 100 μL of cold lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% Nonidet P40 Substitute, 0.01% digitonin and 1% BSA), with a cell concentration of 100,000 to 300,000 cells per 100 μL of lysis buffer. After centrifugation to remove the supernatant, the cells were resuspended in 1× dilution buffer (10x Genomics; 2000207). 5 µl cell suspension was mixed 1:1 with trypan blue for counting using a Countess II FL automated cell counter. Finally, 20,000 to 30,000 cells were used for mtscATAC-seq library preparation. scATAC-seq libraries were prepared following the manufacturer protocol of Chromium Next GEM Single Cell ATAC Kit v2 (10x Genomics, PN-1000390). Briefly, the required number of cells were incubated in a Transposition Mix that includes ATAC Buffer B and ATAC Enzyme at 37 °C for 30 min. Subsequently, a pre-mix comprising Barcoding Reagent B, Reducing Agent B, and Barcoding Enzyme, along with the transposed cells, Single Cell ATAC Gel Beads, and partitioning oil, was loaded onto a Chromium Chip E. The resulting single-cell GEMs were collected, and linear amplification was performed in a C1000 Touch Thermal Cycler with the following conditions: 72°C for 5 minutes, 98°C for 30 seconds, followed by 12 cycles of 98°C for 10 seconds, 59°C for 30 seconds, and 72°C for 1 minute. Emulsions were then coalesced using the Recovery Agent and purified with Dyna beads. Indexed sequencing libraries were constructed, purified, and sequenced on an Illumina-x-plus instrument at Novogene Biotechnology Co., Ltd. (Beijing, China). mtscATAC-seq