GSM8487359: Adapter_free_MT3; Mus musculus; ATAC-seq
The myotube cells were permeabilized in 50 μL of detergent-added ATAC buffer (10 mM Tris-HCl at pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL, 0.1% Tween-20, 0.01% digitonin), followed by a 3-min incubation on ice. One milliliter of ice-cold resuspension buffer with Tween-20 (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20) was added, and tubes were inverted three times to mix. The cell suspension was further passed through a BD insulin needle once to completely lyse the cells and release the nuclei. The density of the resulting nuclear suspension was calculated by hemocytometer counting. A volume corresponding to ∼50,000 nuclei was transferred to a new LoBind tube and spun at 500×g for 10 min at 4°C to pellet nuclei. After carefully removing the supernatant, the nuclear pellet was resuspended in 50 μL of transposition mixture (Illumina Tagment DNA enzyme and buffer small kit 20034197) with 0.01% digitonin and 0.1% Tween-20 and incubated for 30 min at 37°C in a thermomixer with shaking at 1000 rpm. The tagmented chromatin DNA was purified using Minelute Qiagen cleanup kit (Qiagen 28206), and then subjected to PCR-based indexing and library amplification using NEBNext high-fidelity 2× PCR master mix (NEB M0541). The amplified libraries were size-selected using Pippin Prep (2% gel cassette for 100- to 600-bp fragments) and purified using AMPure XP beads (Beckman Coulter A63880).