GSM7651204: ATAC-Seq SRR2-18_47D_biol_rep2; Mus musculus; ATAC-seq
30,000 ESC-derived NSPCs were dissociated with Accutase and washed once with 0.04% BSA in PBS. Cells were lysed in cold resuspension buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2) supplemented with 0.1% NP-40, 0.1% Tween-20 and 0.01% digitonin for three minutes. Cell lysis was halted by dilution with 0.1% Tween-20 in resuspension buffer before pelleting nuclei at 500 RCF for 10 minutes in a 4 °C microcentrifuge. Isolated nuclei were resuspended in a 50 μL transposition mix (25 μL of 2X Tagment DNA buffer [20 mM Tris-HCl, 10 mM MgCl2, 20% dimethyl formamide], 2.5 μL transposase (100 nM final), 16.5 μL PBS, 0.5 μL 1% digitonin, 0.5 μL 10% Tween-20, and 5 μL water) and incubated at 37°C for 30 minutes in a thermomixer, with shaking at 1000 RPM. Transposed fragments were cleaned up with the MinElute kit (Quiagen). Libraries were amplified for 11-14 cycles with universal and indexing primers using NEBNext HiFi 2X PCR Master Mix and dual size selection with 0.7X and 0.3X volumes of AMPure XP beads (Beckman Coulter). Library size was assessed using the 2100 Bioanalyzer (Agilent) before NovaSeq 6000 sequencing using paired-end 50 cycle parameters at a depth of 50-60 million reads per sample.