title: GSM8485693: ATAC, 10K, mito, N2B27, rep1; Mus musculus; ATAC-seq
library_construction_protocol: Briefly, 50,000 cells were sorted or collected after trypsinization. Cells were then pelleted, washed once with cold PBS, and kept on ice. Subsequently, cells were resuspended in 50 µl cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40), and centrifuged. For the transposition reaction, pelleted nuclei were resuspended in 50 µl of 0.5 µM Tn5 (in-house produced according to (Chen et al., 2017)) in TAPS-DMF buffer (10  mM TAPS-NaOH, 5 mM Mgcl2, 10% DMF) and incubated at 37°C for 30 min. During the reaction, the tubes were flicked gently several times, and DNA was immediately purified using the DNA Clean & Concentrator kit (Zymo Research, D4003) with a binding buffer to sample ratio of  5:1. Transposed DNA was diluted in 10 µl DNA elution buffer, and amplified in a solution containing 0.5 mM Ad1.1 universal primer, 0.5 mM of Ad2.X indexing primer, 0.6X SYBR Green I and 1X NEBNext High-Fidelity 2X PCR Master Mix at 72°C for 5 min, 98°C for 30 s, and 5 cycles of 98°C for 10 s, 63°C for 30 s, and 72°C for 1 min. The PCR reaction was monitored to stop the amplification before saturation by sampling 10 µl of amplified DNA and analyzing it by qPCR. The number of cycles to add was determined by the time at which the fluorescence was still in the rising phase. The remaining DNA was further amplified at 98°C for 30 s, and 4-7 cycles of 98°C for 10 s, 63°C for 30 s, and 72°C for 1 min. Next, the library was purified using the DNA Clean & Concentrator kit with a binding buffer-to-sample ratio of 5:1. The purified library was further size-selected using a 0.55X and 1.2X ratio of AMPure XP beads. Library concentrations were determined by Qubit, analyzed on a fragment analyzer, and sequenced on an Illumina NextSeq 500 using 75 nucleotide read-length paired-end sequencing. Libraries were prepared using NEBNext High-Fidelity 2X PCR Master Mix