Ab5131

Chromatin IP was performed as described in [57 (link)], and 3–5 independent biological replicates were produced for each antibody. A dounce homogenizer was used to grind 2–4h old wild-type or Cdk9 Drosophila embryos and a Diagenode Bioruptor was used to sonicate the material before IP. The antibodies used were α-Pol II CTD (8 μl, 8WG16, Abcam ab817), α-Pol II Ser2 (7 μl, Abcam ab5095), α-Ser5 (5 μl, Abcam ab5131). Twenty-five or 30 μl Protein A/G-coated magnetic bead mix slurry and 40–50 μl of embryos were used for each IP. ChIP samples were eluted in 160 μl 0.1x TE pH 8 and duplicates with 2 μl DNA each analyzed by quantitative PCR using a Bio-Rad CFX96 machine and HOT FIREPol EvaGreen master mix (Solis BioDyne). From each IP, the percent of input signal was obtained using the average of the two duplicates, and the Ser5/CTD and Ser2/CTD ratios calculated using these values. Pol II occupancy and Pol II 5’/3’ ratios were calculated by normalizing CTD percent input signal against an intergenic locus. Student´s unpaired t-test for two-sample equal variance was used to find statistically significant differences (p<0.05) between control and Cdk9 samples. All values and calculations can be found in S4 Table.

The preparation of ChIP and input DNA libraries were performed as previously described [27 (link)]. Briefly, cells were crosslinked with 1 % formaldehyde for 10 min at room temperature and quenched with 125 mM glycine. The fragmented chromatin fragments were pre-cleared and then immunoprecipitated with Protein A + G Magnetic beads coupled with anti-H3K4me1 (ab8895, Abcam), anti-H3K4me3 (ab8580, Abcam), anti-H3K27ac (ab4729, Abcam), anti-H3K27me3 (07–449, Millipore), and anti-RNA Pol II (ab5131, Abcam) antibodies. After reverse crosslinking, ChIP and input DNA fragments were end-repaired and A-tailed using the NEBNext End Repair/dA-Tailing Module (E7442, NEB) followed by adaptor ligation with the NEBNext Ultra Ligation Module (E7445, NEB). The DNA libraries were amplified for 15 cycles and subjected to deep sequencing with an Illumina HiSeq 2000.

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.

ChIP was performed as described previously^{89 (link)}. Anti-JMJD3 antibody (AP1022a, Abgent), anti-NICD (4147, Cell Signaling), anti-trimethyl H3K27 antibody (07–449, Millipore), anti-monomethyl H3K27 antibody (07–448, Millipore), anti-phosphorylated RNA polymerase II antibody (ab5131, Abcam), anti-p50 antibody (ab7971, Abcam), anti-p65 antibody (ab7970, Abcam), or normal IgG (SC-2027, Santa Cruz Biotechnology) were used. Real-time PCR was performed with a Stratagene Mx3000P using primers (see Supplementary Table S2).