title: GSM8853807: bulkATAC-Seq, Handling_0min_CRS3_v49; Mus musculus; ATAC-seq
library_construction_protocol: Nuclei isolation was performed as described in Grandi et al., 2022. All steps were performed on ice. Briefly, one vHC was homogenized in lysis buffer (26 mM Sucrose, 30 mM KCl, 10 mM MgCl2, 20 mM Tricine-KOH (pH 7.8), 1 mM DTT, 0.5 mM Spermidine, 0.15 mM Spermine, 0.3% NP-40, Complete protease inhibitor in nuclease-free water) by 10 strokes with pestle A and 20 strokes with pestle B. The sample was filtered through a 70 μm Flowmi strainer and centrifuged for 5 min at 400 g at 4°C. Supernatant was removed and additional lysis buffer was added to resuspend the pellet. The sample was then mixed with 50% Iodixanol solution (30 mM KCl, 26 mM Sucrose, 10 mM MgCl2, 20mM Tricine-KOH (pH 7.8), 50% Iodixanol solution in nuclease-free water) and underlaid by 30% Iodixanol solution (50% Iodixanol solution in lysis buffer) and 40% Iodixanol (50% Iodixanol solution in lysis buffer) layers. The sample was centrifuged for 20 min at 3000 g at 4°C with brakes off. The nuclei band was collected and mixed with ATAC buffer (10 mM NaCl, 10 mMTris-HCl (pH7.5), 3 mM MgCl2, 0.1% Tween20 in nuclease-free water). The nuclei count was assessed and 50'000 nuclei were transferred to a new tube with ATAC buffer. The samples was centrifuged for 10 min at 500g at 4°C and supernatant was removed. Nuclei were resuspended in 50 μl of transposition mix (25 μl 2× TD buffer, 2.5 μl transposase (Diagenode, C01070012), 16.5 μl PBS, 0.5 μl 1% digitonin, 0.5 μl 10% Tween-20, and 5 μl nuclease-free water). Transposition reactions were incubated at 37 °C for 30 min while shaking at 1,000 r.p.m.. Tagmented DNA was purified using the MinElute Reaction Cleanup Kit (Qiagen, 28204). Purified DNA was amplified using the NEBNext high-Fidelity 2X master mix (New England Biolabs, Ipswich, Massachusetts, United States, M0541) and Diagenode primers (Diagenode, Marlborough, Massachusetts, United States, C01011036) 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. A fraction of the amplification product was used to determine the number of additional cycles using qPCR, resulting in a total of 8-9 amplification cycles for all samples. A double-sided size selection (0.5X and 1.3X) was performed to remove large and very small library fragments. Insert size of the library was validated and quantified on an Agilent 2100 Bioanalyzer.