GSM5849878: Suv39hdn_Asynchr_1; Mus musculus; ATAC-seq
For ATAC-seq on asynchronous cells, nuclei were obtained by resuspending 5 x 104 cells in 50 μl of ATAC-resuspension buffer (10 mM Tris–HCl, pH 7.4; 10 mM NaCl; 3 mM MgCl2; 0.1% Igepal CA-630; 0.1% Tween-20; 0.01% Digitonin) for 3 min on ice before adding 1 ml of ATAC-resuspension buffer without Igepal and Digitonin and pelleting nuclei at 500 g (10 min at 4°C). For ATAC-seq on purified mitotic chromosomes, condensed chromosomes were released from arrested cells using polyamine buffer, stained with Hoechst 33258 and Chromomycin A3, purified by flow cytometry using a Becton Dickinson Influx equipped with specialised air-cooled lasers and pelleted at 10,000 g (10 min at 4°C). To perform ATAC-seq, nuclei, mitotic cells (5 x 10^4) or purified chromosomes (2 x 10^6) were resuspended in 50 μl transposase mixture (25 μl Illumina TD buffer, 22.5 μl H2O, and 2.5 μl Illumina TDE1 transposase) and incubated at 37°C for 30 min with shaking at 1,000 rpm. DNA was purified (Qiagen MinElute kit) and amplified with seven cycles of PCR (NEBNext High Fidelity master mix). Libraries were purified twice with Ampure XP beads (Beckman Coulter), including removal of large fragments with 0.5x beads.