Chromatin Immunoprecipitation and Sequencing

Nuclei were isolated from cross-linked samples as described previously^{50 (link)} and resuspended in nuclei lysis buffer (50 mM Tris-HCl pH8, 10 mM EDTA, 1% sodium dodecyl sulfate (SDS), 1 mM phenylmethanesulfonylfluoride (PMSF), 1% Plant Protease Inhibitors from Sigma). After fragmentation using a Covaris S200, the chromatin samples were diluted with ChIP dilution buffer (final concentration: 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH8.0, 150 mM NaCl, 1 mM PMSF, 0.1% SDS, 1% Plant Protease Inhibitors, Sigma). The diluted chromatin samples were subjected to immunoprecipitation with antibodies (anti-MYC tag, clone 4A6, Millipore 05–724 (30 μg); Anti-RNA polymerase II CTD repeat YSPTSPS antibody [8WG16] Abacm ab817 (20 μg); Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody, Abcam ab5095 (30 μg); Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody Abcam ab5131 (30 μg); and control IgG Abcam ab18413 (20 μg)).
Native histone ChIP was performed as described previously^{51 (link)} using anti-Histone H3 Abcam ab1791 (10 μg).
The ChIP libraries were prepared using the NEBNext® Ultra™ DNA Library Prep Kit (New England Biolabs) and then sequenced on a NextSeq 500 (Illumina) at the Center of Genomics and Bioinformatics, Indiana University. All high-throughput sequencing data and corresponding experimental details are available in GEO SuperSeries GSE112443.