bulk ATAC of mus musculus: fancm wildtype sperm
Bulk tagmentation was performed using either commercial pre-assembled Tn5 transposase (Diagenode) or with home-made Tn5 transposases produced and assembled using previously published methods (Picelli et al., 2014). Approximately 50,000 haploid sperm nuclei were FACS sorted into a 5 mL polypropylene tube containing 300 uL NWRB with 0.1% (w/v) BSA. Nuclei were centrifuged at 500  g for 5 minutes at 4oC and the supernatant was aspirated. Without disrupting the pellet, 1 mL of OMNI-ATAC RSB buffer (10 mM TrisHCl pH 8.0, 10 mM NaCl, 3 mM MgCl2) was slowly added and the sample was incubated for 5 minutes, then resuspended gently using a wide bore tip. Nuclei were centrifuged at 500  g for 5 minutes at 4oC and the supernatant was aspirated. Nuclei were resuspended in 100 L chilled OMNI-ATAC RSB buffer containing 0.1% (v/v) Tween 20, 0.1% (v/v) IGEPAL, and 0.01% (v/v) Digitonin and incubated on ice for 2 minutes. Nuclei were centrifuged again at 500  g for 5 minutes at 4oC and the supernatant was aspirated. Nuclei were resuspended in 50 L freshly prepared transposition mix (33 mM TrisHCl pH 8.0, 66 mM Potassium Acetate, 10 mM Magnesium Acetate, 15% (v/v) N,N-dimethylformamide, 0.01% (v/v) Digitonin, 2.5 L Tn5 assembled enzyme) and mixed by pipetting up and down ten times. Transposition reactions were incubated at 37C for 45 minutes in a thermocycler with gentle mixing by tapping the tube at 5-minute intervals. Transposition reaction was stopped with 50 L 2xATAC-STOP buffer (10 mM TrisHCl pH 8.0, 20 mM EDTA). Afterwards, the samples were purified using a Monarch Nuclei Acid Purification Kit (NEB) as per manufacturers instructions and eluted in 20 L of nuclease free water. All libraries were amplified with barcoded primers listed below, using previously described methods (Buenrostro et al., 2015).  Each library was sequenced on the NovaSeq 6000 (Illumina) using a 50/8/16/50 bp read configuration. Tagementation was used for library selection.