title: GSM9350821: Skeletal Muscle, Female D2W-1, snATAC-Seq; Mus musculus; ATAC-seq
library_name: GSM9350821
library_construction_protocol: Frozen tibialis anterior muscles were cryosectioned on dry ice and homogenized using a gentleMACS Dissociator (Miltenyi; “Protein_01_01” program) in MACS buffer consisting of 5 mM CaCl₂, 2 mM EDTA, protease inhibitor (Roche), 3 mM magnesium acetate, 10 mM Tris-HCl (pH 8.0), and 0.6 mM DTT. The homogenate was passed through a 30 μm CellTrics filter (Sysmex) and collected in 15 ml tubes. Nuclei were pelleted by centrifugation at 500 × g for 5 min at 4°C, resuspended in Nuclear Permeabilization Buffer (PBS containing 5% BSA, 0.2% IGEPAL CA-630, 1 mM DTT, and protease inhibitors), and incubated for 5 min at 4°C on a rotating platform. After a second centrifugation at the same settings, nuclei were transferred into a high-salt tagmentation buffer (36.3 mM Tris-acetate, 72.6 mM potassium acetate, 11 mM magnesium acetate, and 17.6% DMF), counted with a hemocytometer, and diluted to a concentration of 1,000 nuclei in 9 μl. Aliquots of 1,000 nuclei were dispensed into four 96-well plates (384 wells total). Barcoded Tn5 transposomes (1 μl per well) were added using a BenchSmart 96 (Mettler Toledo), and tagmentation was carried out at 37°C for 90 min with shaking at 900 rpm. The reaction was terminated with 2.5 μl of 100 mM EDTA (20 mM final) followed by an additional 15 min incubation at 37°C. Each well then received 6 μl of 3× sort buffer (PBS supplemented with 3% BSA and 3 mM EDTA). All wells were pooled, stained with Draq7 (1:500; Cell Signaling), and sorted on a Sony SH800. Eighty nuclei were deposited per well into four new 96-well plates containing 8 μl of primer mix (15 pmol i7, 15 pmol i5, 200 ng BSA, and 0.027% SDS). Plates were incubated at 65°C for 25 min, followed by the addition of 4 μl of 1.33% Triton X-100 to neutralize SDS. Each well received 11 μl of Q5 PCR master mix (5 μl 5× buffer, 0.5 μl 10 mM dNTPs, 0.25 μl Q5 polymerase, and 5.25 μl water), and amplification was performed with the following cycling parameters: 72°C for 4 min; 98°C for 30 s; 12 cycles of 98°C for 10 s, 60°C for 30 s, and 72°C for 45 s; and a final hold at 4°C. PCR products were pooled and purified using the Zymo DNA Clean & Concentrator-5 kit. Fragments between 200–600 bp were size-selected on a 1.5% agarose gel and recovered with the Zymo Gel DNA Recovery kit. Library concentration was measured with the Qubit 1× dsDNA HS assay (Invitrogen), and fragment size distribution and nucleosomal periodicity were evaluated using a High Sensitivity D1000 TapeStation (Agilent). Next-generation sequencing of snATAC libraries was performed on the DNBSEQ G400 platform using paired-end sequencing, 50-bp reads for each end, generating approximately 5,000-10,000 raw reads per cell