GSM7011847: H3K27ac CUT&Tag KO with GSK126 treatment rep 2; Mus musculus; OTHER
Beads (with bound nuclei) were magnetized, supernatants were removed, beads were washed once with 400 ìl antibody buffer (Wash buffer supplemented with 2 mM EDTA and 0.05% Digitonin), and resuspended in 200 ìl antibody buffer containing the primary antibody in 1:100 v/v dilution (H3K27me3 - rabbit monoclonal, Cell Signaling Technology, cat. no. 9733). Primary antibody incubations were performed on a rotating platform overnight at 4oC. Beads were magnetized, supernatant removed, washed once with 400 ìl Dig-Wash (Wash buffer supplemented with 0.05% Digitonin), resuspended in 200 ìl Dig-Wash containing guinea pig á-rabbit IgG secondary antibody (Antibodies online cat. no. ABIN101961) at 1:00 dilution. Secondary antibody incubations were performed on a rotating platform for 30 min to 1 hr at room temperature. Beads were magnetized, supernatant removed, washed twice with 400 ìl Dig-Wash to remove unbound antibodies, and resuspended in 200 ìl Dig-Med buffer (Dig-Wash buffer, except contai  ning 300 mM NaCl) with 1:200 dilution (~0.04 ìM) of pA-Tn5 pre-loaded with double-stranded adapters with 19mer mosaic ends (Kaya-Okur et al., 2019). pA-Tn5 incubations were performed on a rotating platform for 1 hr at room temperature. Beads were magnetized, supernatant removed, washed three times with 400 ìl Dig-Med to remove unbound pA-Tn5, and resuspended in 300 ìl Tagmentation buffer (Dig-Med supplemented with 10 mM MgCl2). Tagmentation reactions were performed by incubating samples at 37oC on a rotating platform for 1 hr. Tagmentation reactions were stopped by sequentially adding 10 μl of 0.5 mM EDTA, 3.1 μl of 10% SDS (1% final), and 2 uL of 20 mg/mL Proteinase K, mixed well, and incubated in 50oC water bath for 1 hr. DNA was extracted using phenolchloroform-isoamyl alcohol (25:24:1), re-extracted with one volume CHCl3, aqueous phase (~300 μl) was transferred to fresh tube, and DNA was precipitated by adding 900 μl ethanol overnight at -20°C followed by centrifugation for 45 min at 13,000 rpm and 4°C. Pellets were rinsed with 100% ice-cold ethanol, air-dried, dissolved in 30 μL 0.1 x TE (1 mM Tris pH 8, 0.1 mM EDTA) supplemented with RNase A, and incubated in 37°C water bath for 15 min. To amplify libraries, 21 μL DNA was mixed with 2 μL each of a universal i5 primer and a uniquely barcoded i7 primer, using a different barcode for each sample, and 25 μL of NEBNext HiFi 2x PCR Master mix. Samples were placed in a thermocycler with heated lid using the following cycling conditions: 72°C for 5 min (gap filling); 98°C for 30 sec (denaturation); 13 cycles of 98°C for 10 sec and 63°C for 10 sec (combined annealing and extension); final extension at 72°C for 1 min, and held at 8oC. Post-PCR clean-up was performed using a double-sided clean-up method. 25 μL (0.5 X) of Ampure XP beads (Beckman Counter) was mixed with the libraries and incubated for 10 min at room temperature to remove large DNA fragments (>1300 bp). Beads were magnetized, supernatants were removed to fresh tubes and mixed with 45 μL (1.4 X - 0.5 X = 0.9 X) of Ampure XP beads and incubated for 10 min at room temperature. Beads were magnetized and the supernatants containing unused PCR primers were discarded. Beads were washed three times with 80% ethanol and eluted in 30 μL 10 mM Tris pH 8.0. Libraries were sequenced for 25 cycles in 25bp paired-end mode on the Illumina HiSeq 2500 platform at the Fred Hutchinson Cancer Research Center Genomics Shared Resource. The detailed, step-by-step original protocol can be found at https://www.protocols.io/view/bench-top-cut-amp-tag-kqdg34qdpl25/v3 PCR amplification using indexed primers PMID: 26083756