title: GSM5788470: MYC-DMSO; Homo sapiens; OTHER
library_construction_protocol: CUT and RUN was performed with a CUT&RUN assay kit according to the vendor's specifications (Cell Signaling Technology, 86652). Following proteasome inhibition for 3 h with 25 µM Lactacystin or 0.1% v/v DMSO control, cells were collected in conical tubes, counted, and assessed for cell viability. 250,000 MM.1S cells were used per condition and lysed in 4% digitonin. Capturing of DNA/protein complexes was performed with the following antibodies: 2 µl of anti-MYC antibody (Cell Signaling Technology, 13987) per condition, 2 µl of anti-FLAG antibody (Sigma-Aldrich, Clone M2, F1804) per condition for MM.1S cells stably transduced with 3xFLAG-ubiquitin, and as a negative control 5 µl of rabbit IgG isotype control (Cell Signaling Technology, 66362) per condition. Single-indexed DNA libraries were prepared with the Ultra Next DNA library prep kit II (NEB, E7645S) and multiplexed for sequencing using NEBNext Multiplex oligos (E7500S). The protocol for DNA library preparation was adapted from previous publications and specifically optimized for CUT and RUN samples (Skene P.J. & Henikoff S., eLife, 2016, PMID 28079019; Liu N. et al., Cell, 2018, PMID 29606353). Library quality control, including assessment of fragment size distribution and quantification by qPCR, and library sequencing was conducted by the Baylor College of Medicine Genomic and RNA Profiling Core. The size distribution of the final libraries was confirmed by electrophoretic analysis on an Agilent Bioanalyzer 2100 prior to sequencing. Pooled libraries were loaded onto a NextSeq 500 high output v2.5 flow cell (Illumina, 20024906) and analyzed on a Illumina NextSeq 500 sequencing system. The flow cell was sequenced in a 75 bp paired-end run, enabling the generation of a minimum of 19 million reads per sample. Clustering and sequencing performance were controlled as previously described (Sweeney M.A. et al., Sci. Rep, 2020, PMID 32811853).