GSM9135782: Msx2-KO, dorsal skin, hair follicle stem cells, 6-month-old telogen, ATAC-seq, rep 1; Mus musculus; ATAC-seq
Omni-ATAC-seq was performed following the published protocol (PMID: 28846090) with minor modifications. FACS-isolated HFSCs from different genetic backgrounds were resuspended in lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% NP-40, 0.1% Tween-20, and 0.01% Digitonin) and incubated for 5 minutes at 4 °C. Following lysis, nuclei were diluted by adding ATAC-resuspension buffer (identical to lysis buffer but lacking NP-40 and Digitonin, and supplemented with 0.1% Tween-20). Nuclei were pelleted by centrifugation at 2,500 rpm for 5 minutes at 4 °C. The nuclear pellet was then resuspended in the transposition reaction mix containing Tn5 transposase (Illumina, 15027866) and incubated at 37 °C for 30 minutes. Transposed DNA was purified using the Zymo DNA Clean & Concentrator Kit (D4014) and eluted according to the manufacturer's instructions. Library amplification was performed based on the published Omni-ATAC-seq protocol (PMID: 28846090). Samples were PCR-amplified using NEBNext High-Fidelity PCR Master Mix (M0541, New England Biolabs) and 1.25 μM custom Nextera primers under the following thermal cycling conditions: 72 °C for 5 min, 98 °C for 30 s, followed by 5 cycles of 98 °C for 10 s, 63 °C for 30 s, and 72 °C for 1 min. After the initial 5 cycles, a 5 μl aliquot was removed, combined with SYBR Green I (Invitrogen, S7563), and subjected to quantitative PCR (qPCR) to determine the optimal number of additional cycles. The final number of cycles was defined based on reaching one-fourth of the maximum fluorescence intensity. Libraries were then amplified accordingly and purified. Final DNA was eluted in 15 μl of 10 mM Tris-HCl (pH 8.0).