GSM8746942: BM Eosinophil_PBS2; Mus musculus; ATAC-seq
Nuclei were isolated from a total of 80 000 viable bone marrow derived eosinophils through lysis. Briefly, cells were pelleted at 500 g for 5 minutes at 4°C prior to gentle, but thorough resuspension in 50 µl ATAC-seq resuspension buffer (10 mM Tris-HCL, 10 mM NaCl and 3 mM MgCl2 in molecular biology grade H2O) supplemented with 0.1% NP-40 (Sigma #I8896), 0.1% Tween 20  (Sigma #9005-64-5) and 0.01% digitonin (Sigma #D141). Cells were then incubated at 4°C for 3 minutes, followed by addition of 1 ml cold ATAC-seq resuspension buffer with only 0.1% Tween 20. Nuclei were then pelleted by centrifugation for 10 minutes at 500 g and 4°C. The pellet was resuspended with 50 µl of tagmentation mixture containing 25 µl of 2X tagmentation buffer (Diagenode #C01019043), 2 µl of tagmentase (Diagenode #C0170012), 1 µl of 50x EDTA-free protease inhibitor (Roche/Sigma, #11836170001), 0.5 µl of 1% digitonin, 2.5 µl 1% NP-40 and 19 µl H2O and incubated at 37°C for 30 minutes. After tagmentation, DNA was purified using the MiniElute PCR purification Kit (Qiagen). The protocol was adapted from a previously published protocol (J. M. Felton et al., Epigenetic Analysis of the Chromatin Landscape Identifies a Repertoire of Murine Eosinophil-Specific PU.1-Bound Enhancers. The Journal of Immunology 207, 1044–1054 (2021),  DOI: 10.4049/jimmunol.2000207). Libraries were constructed according to the OMNI-ATAC protocol (Felton et al., 2021, DOI: 10.4049/jimmunol.2000207 and Corces et al, 2017, DOI: 10.1038/nmeth.4396 ). Quality was assessed with an Agilent Bioanalyzer and libraries were sequenced on the Illumina Novaseq 6000, paired-end, 150 bases each end.