GSM8404620: U2OS H3K9ac C207 treatment Rep2; Homo sapiens; OTHER
The procedure for the isolation of nuclei and subsequent CUT&RUN of AAV genomes was adapted from the Epicypher CUTANA protocol (https://www.epicypher.com/resources/protocols/cutana-cut-and-run-protocol; see especially Appendex I: Isolation of Nuclei) using BioMag Plus Concanavalin A beads (Polysciences 86057), pAG-MNase (Cell Signaling Technologies 40366), and primary antibodies for BRD4 (Abcam ab128874, 1:50) or IgG (Jackson ImmunoResearch 009-000-003, 1:50). Approximately 500k U2OS nuclei were used for each CUT&RUN reaction. U2OS cells were permeabilized in the presence of 0.01% digitonin. Primary antibody incubation occurred on a nutating mixer (Corning 10-320-100) at 4C overnight. To improve the signal-to-noise ratio, an additional step of secondary antibody incubation (Guinea Pig Anti-IgG, ABIN101961, 1:100) on a nutating mixer for 1h at 4C was included. Per reaction, 50 ng of S. cerevisiae genomic spike-in DNA (Provided with Cell Signaling Technologies 40366) was added with the addition of stop buffer. Released DNA fragments were purified using the DNA Clean & Concentrator-25 Kit (Zymo D4033) and double eluted in 28 uL of warm elution buffer. CUT&RUN-enriched DNA was barcoded and amplified using the NEBNext Ultra II DNA Library Prep with Sample Purification Beads kit (New England Biolabs E7103L) and NEBNext Multiplex Oligos for Illumina (E6440L). Bead based size selection was conducted by a 28 uL first bead addition and a 13 uL second bead addition. Library concentrations were determined using the Qubit 1X dsDNA High Sensitivity (HS) Assay Kit (Invitrogen Q33230) and a Qubit 4 Fluorometer (Thermo Fischer). The average fragment size of each library (~350-450 bp) was determined using High Sensitivity D1000 Reagents (Agilent 5067-5585) and ScreenTape (Agilent 5067-5584) on a 2100 Bioanalyzer (Agilent). Libraries were pooled and sequenced to a depth of > 11M reads per sample (2 x 150 bp paired-end reads) with NextSeq1000/2000 P2 Reagents 300 cycles (Illumina 20050264).