title: GSM8250303: Mitotic chr, PBK KO, rep1; Mus musculus; ATAC-seq
library_construction_protocol: Mitotic chromosomes were extracted and purified by flow cytometry. Briefly, arrested preB cells (~108) were incubated for 20 min at RT in 10 ml of hypotonic solution (75 mM KCl, 10 mM MgSO4, 0.5 mM spermidine trihydrochloride (Sigma-Aldrich, S2501), 0.2 mM spermine tetrahydrochloride (Sigma-Aldrich, S2876), pH 8.0), followed by 15 min on ice in 3 ml of polyamine buffer (80 mM KCl, 15 mM Tris-HCl, 2 mM EDTA, 0.5 mM EGTA, 3 mM DTT, 0.25% Triton X-100, 0.5 mM spermidine trihydrochloride, 0.2 mM spermine tetrahydrochloride, pH 7.7). Samples were vortexed for 30 s at maximum speed, passed several times through a 21-gauge needle, centrifuged at 200 g for 2 min, filtered through a 20 μm CellTrics filter (Sysmex) and stored at 4 °C overnight. Next day, chromosomes were stained on ice by adding 5 μg/ml Hoechst 33258 (Sigma-Aldrich, 94403), 25 μg/ml Chromomycin A3 (Sigma-Aldrich, C2659) and MgSO4 (10 mM final) for 45 min, followed by addition of sodium citrate (10 mM final) and sodium sulfite (25 mM final) for 1 h.  Stained chromosomes were purified using a Becton Dickinson Influx with BD FACS Sortware (v1.2.0.142), using a 70 μm nozzle tip, a drop-drive frequency of 96 kHz and a sheath pressure of 448 kPa. Forward scatter was measured with a 488 nm laser (Coherent Sapphire, 200 mW); Hoechst 33258 was analysed with a 355 nm air-cooled laser (Spectra Physics Vanguard, 350 mW) and 400 nm (long-pass)/500 nm (short-pass) filters; Chromomycin A3 was analysed with a water-cooled 460 nm laser (Coherent Genesis, 500 mW) and 500 nm (long-pass)/600 nm (short-pass) filters. 2 x 106 flow-sorted chromosomes were pelleted at 10,000 g for 10 min at 4 °C before tagmentation in a 50 μl reaction (2.5 μl Tagmentase (loaded Tn5 transposase, Diagenode, C01070012), 25 μl 2X Tagmentation buffer (Diagenode, C01019043), 22.5 μl H2O) at 37 °C for 30 min with 1,000 rpm shaking. DNA was purified (Qiagen MinElute PCR purification kit) and PCR amplified (7 cycles; NEBNext High Fidelity master mix) with primers containing sequencing adapters. Amplified libraries were purified using 1.8X volume Ampure XP beads (Beckman Coulter), firstly using 0.5X beads to remove large fragments, before using the remaining volume of beads to capture and purify libraries.