GSM4630470: ATAC_Late_rep1; Mus musculus; ATAC-seq
Hippocampal tissue was extracted and flash frozen. After extraction, samples were immediately homogenized in 0.5 mL ice cold PBS with protease inhibitors (Pi) (11836170001, Roche). The suspension was centrifuged at 1200 g, 4°C, for 5 minutes and the pellet was resuspended in 5 mL NF-1 hypotonic buffer (0.5% Triton X-100/0.1M Sucrose/5mM MgCl2/1mM EDTA/10mM Tris-HCl, pH 8.0, Pi). Pelleted nuclei were resuspended in and incubated with NeuN, Arc and GFP primary antibodies for overnight in 4°C. Unbound antibodies were washed out twice and nuclei were filtered with 40 µm mesh filter for FACS sorting. ATAC seq libraries were prepared as previously described in Buenrostro, J. D, et al, Nat. Methods, 2013, with minor modification. Immediately follow sorting, cells were spun down (4°C, 1,600 x g for 15 min) and resuspended in fresh 50 ul cold lysis buffer (0.15% NP-40 instead of Igpal) and rocked on ice for 10 min. Next, tagmentation reaction was carried out in 25 ul volume (12.5 μl TD, 1.25 μl TDE1, 11.25 μl nuclease-free water), for 30 min at 37c with gentle rocking. Tagmented DNA fragments were amplified and barcoded using PCR reaction (1 cycle; 5min – 72c, 30sec – 98c, 10 cycles; 15sec – 98c, 30sec – 60c, 3min – 72c). Aamplified DNA was cleaned and purified using 1.8x AMPure beads.
GEO Accession
GSM4630470