GSM6597824: DLD-1 cln18 H3K27ac biol rep 1; Homo sapiens; OTHER
CUTANA CUT&RUN, Illumina sequencing, and data analysis. CUTANA(R) CUT&RUN was performed on an automated protocol (autoCUT&RUN) derived from those previously described [1-3]. In brief, for each CUT&RUN reaction 500K cells [5 million cells/mL prepared in 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 mM Trichostatin A, 1X EDTA/EGTA-free Complete Protease Inhibitor] were dispensed to individual wells of a 96-well plate, immobilized onto Concanavalin-A beads (Con-A; EpiCypher #21-1401), and incubated overnight (4°C) with 0.5 µg of antibody (IgG, H3K4me3, H3K27me3, H3K27ac) (all antibodies validated to histone post-translation modification (PTM)-defined SNAP-ChIP nucleosome standards as previously [4]). pAG-MNase (EpiCypher #15-1016) was added / activated (2 hours @ 4°C) and CUT&RUN enriched DNA purified using Serapure beads after mixing at 2:1 (Bead:DNA) ratio. Recovered DNA was quantified using PicoGreen and reactions were normalized to 5ng DNA (or entirety of the reaction if <5ng DNA was recovered) before preparing sequencing libraries (CUTANA CUT&RUN Library Prep kit;EpiCypher #14-1001). All autoCUT&RUN steps were optimized / performed on Tecan Freedom EVO robotics platforms with gentle rocking for incubation steps and magnetic capture for media exchange / washing steps.  •        [1] Marunde et al (2022) bioRxiv [doi.org/10.1101/2022.02.21.481373]  •        [2] Yusufova et al (2021) Nature 589:299 [PMID: 33299181]   •        [3] Skene et al (2018) Nat Protoc 13:1006  •        [4] Shah et al (2018) Molecular Cell 72:172 [PMID: 30244833] Library was prepared according to Epicypher CUT&RUN Kit library prep manual https://www.epicypher.com/content/documents/manuals/14-1001-2-cut-and-run-library-prep-kit-manual.pdf