GSM7311610: INT-2_S2; Ambystoma mexicanum; ATAC-seq
Cells were dissociated in dissociation buffer (100 mg/ml liberase diluted in 0.7X PBS with 0.5 U/ml DNase I) for 30 min at room temperature with gentle mixing. Cells were then stained with 1 mg/ml Hoecsht, calcein AM, and propidium iodide for isolation of alive, dividing (4C) and non-dividing (2C) cells by fluorescence activated cell sorting. The 4C cells were used for library preparation. Ten thousand sorted 4C cells from homeostatic, systemically-activated, and regenerating limbs were resuspended in 5 μL PBS. 42.5 μL of transposition buffer (38.8mM tris-acetate, 77.6mM potassium acetate, 11.8mM magnesium acetate, 0.12% NP-40, 18.8% DMF, 0.47% protease inhibitor cocktail) was added to the cells, mixed, and incubated 10 min at room temperature. 2.5 μL of pre-loaded Tn5 (Diagenode cat# C01070012) was added to the reaction mixture. The transposition reaction was carried out at 37°C for 30 min with shaking at 300 rpm. Following transposition, the samples were purified through Direct-zol RNA MiniPrep kit (Zymo Research cat# R2050). Purified library fragments were amplified with custom primers 1 and 2 using the following conditions: 72°C for 5min; 98°C for 30s; and thermocycling at 98°C for 10s, 72°C for 30s and 72°C for 1min. The libraries were amplified for five cycles. Soon after, 5μL aliquot of the PCR reaction was added with 10μL of the PCR cocktail with SYBR Green (0.6Xfinal concentration). 20 cycles of PCR was performed to determine the additional number of cycles needed for the remaining 45 μL reaction. The libraries were purified using Direct-zol RNA MiniPrep kit. NovaSeq S4 2 x 100 bp lane