GSM7843032: Retina, H3K27ac_P14_OIR_rep1; Mus musculus; OTHER
Freshly isolated retinas from three mice were minced and pooled as a group sample. The pooled retina was incubated in NE1 buffer (20 mM HEPES pH7.8, 10 mM KCl, 0.5 mM spermidine, 0.1% Triton X-100, 20% glycerol, Roche proteinase inhibitor) for 10 min on ice, then homogenized by Dounce homogenizer. The suspension was centrifuged at 100 x g for 30 sec at 4°C. The supernatant containing nuclei was transferred to a fresh tube. The nuclei number was counted by a hemacytometer. A half million nuclei were conjugated to activated concanavalin A-coated magnetic beads (Bangs Laboratories, BP531). The beads were separated by a magnet stand and resuspended in Wash buffer (20 mM HEPES pH7.5, 150 mM NaCl, 2.0 mM EDTA, 0.5 mM spermidine, 0.01% digitonin, Roche proteinase inhibitor) with primary antibody (1:50 dilution). After incubation at 4°C overnight on a rotator, the beads were separated, resuspended in Wash buffer with secondary antibody, guinea pig anti-rabbit antibody (1:100 dilution), and incubated for 30 min at RT on a rotator. The beads were washed with Wash buffer once and resuspended in 300-Wash buffer (20 mM HEPES pH 7.5 300 mM NaCl, 0.5 mM spermidine, 0.01% digitonin, Roche proteinase inhibitor) with pAG-Tn5 (EpiCypher, 15-1017). After incubation for 1 hour at RT, the beads were washed with 300-Wash buffer while the beads were on a magnet stand. For tagmentation of DNA, the beads were resuspended in Tagmentation buffer (300-Wash buffer with 10 mM MgCl2) and incubated for 1 hour at 37°C. The tagmentation was eliminated by adding 0.5 M EDTA, 10% SDS, and 20 µg/µl proteinase K (final concentration 16 mM EDTA, 0.1% SDS, 0.16 µg/µl proteinase K) with incubation for 10 min at 70°C. The tagmented DNA was extracted by phenol-chloroform extraction, precipitated by ethanol precipitation, and dissolved in 21 µl TE buffer (1 mM Tris pH8.0, 0.1 mM EDTA). To prepare CUT&Tag libraries, 2 µl of 10 µM i5 primer, 2 µl of 10 µM barcoded i7 primer, and 25 µl of NEBNext HiFi 2x Master mix (NEB, M0541L)) were added. PCR was performed 12 cycles. Following the manufacturer's instructions, the libraries were purified using AMPure XP bead (Beckman Coulter, A63880).