GSM7316844: CUT&TAG_day 4, control, replicate 1; Mus musculus; OTHER
Four days after stroke onset, brains were removed and sliced in ice-cold artificial cerebrospinal fluid. Peri-infarct area was trimmed and treated with pronase (Sigma-Aldrich) for 75 minutes at room temperature. Enzyme-treated tissue was dissociated by gentle pipetting with a Pasteur pipette. Neuron enriched cell populations (Thy1+/ACSA2-/CD31-/CD45-) were collected by fluorescence-activated cell sorting (BD FACS Aria III). The CUT&Tag library was prepared using a slightly modified Bench top CUT&Tag V.2 protocol described elsewhere (https://www.protocols.io/view/bench-top-cut-amp-tag-kqdg34qdpl25/v2). Isolated neurons were bound with activated concanavalin A-coated magnetic beads and primary antibody (1:50 dilution, anti-histone H3 (citrulline R2+R8+R17) antibody: ab5103, Abcam) After removing primary antibody by wash buffer, the samples were bound with secondary antibody (1:100 dilution, guinea pig anti-rabbit IgG antibody: ABIN101961, antibodies-online.com). After two washes with wash buffer, a 1:200 dilution of the pA-Tn5 adapter complex, which was prepared in Dig-325 buffer (0.025% digitonin, 20 mM HEPES, pH 7.5, 325 mM NaCl, 0.5 mM spermidine, 1:100 protease inhibitor cocktail), was added to the sample and then incubated with pA-Tn5 for 1 hour at room temperature. The samples were placed on a magnet stand to clear and washed three times with Dig-325 buffer. Tagmentation buffer (10 mM MgCl2 in Dig-325 Buffer) was added and incubated at 37°C for 1 hour. To stop tagmentation and solubilize DNA fragments, 2.25 mL of 0.5 M EDTA, 2.75 mL of 10% SDS, and 0.5 mL of 20 mg/mL proteinase K were added to each sample, mixed at full-speed vortex for 2 seconds, and incubated at 50°C for 1 hour. For DNA purification, 122 mL of SPRI select beads was added to each sample, spun quickly, and held for 5 minutes. The tubes were placed on a magnetic stand to clear, the supernatant was discarded, the pellet was washed twice with 200 mL of 80% ethanol, and dried for 2 minutes. Then, 11 uL of TE (1 mM Tris-HCl pH 8.0, 0.1 mM EDTA) was added and allowed to stand for 5 minutes. The tube was placed on a magnetic stand, and the liquid was withdrawn to a fresh tube. For library amplification, 10.5 uL of DNA solution was mixed with 1 uL of i5 and i7 primers (10 mM) each with unique barcodes for each sample, followed by the addition of 12.5 uL of NEBNext HiFi 2× PCR Master Mix (New England BioLabs). Samples were placed in a thermocycler with a heated lid using the following cycling conditions: 72°C for 5 min, 98°C for 30 s; 18 cycles of [98°C 10 s, 63°C 30 s]; 72°C 1 min final extension, held at 4°C. Finally, 25 uL of Low-TE was added to the Post-PCR sample and 25 uL of SPRI select beads was added and incubated for 10 minutes. The supernatant was transferred to a new tube, and 15 uL of SPRI select beads was added. Samples were incubated for 10 min, washed twice with 80% ethanol, and eluted with 12 uL of 10 mM Tris pH 8.0.