Ab817

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 following chemicals were used in this study: flavopiridol (F3055, Sigma-Aldrich), radicicol (R-370, Alomone), sodium salicylate (S3007, Sigma-Aldrich), copper sulphate (102790, Merck).
The following primary antibodies were used in this study: α-mouse RNA polymerase II CTD repeat (Abcam, ab817), α-mouse RNA polymerase II CTD repeat phosphor S5 (Abcam, ab5408), α-rabbit RNA polymerase II CTD repeat phospho S2 (Abcam, ab5095), α-rat RNA polymerase II CTD repeat phosphor S2 (Active Motif, Cat#61083), α-rabbit HSF (gift from John Lis), α-rabbit Polyhomeotic (Paro lab stock), α-rabbit Pleiohomeotic (gift from Judith Kassis), α-rabbit Pleiohomeotic (gift from Jurg Muller), α-mouse FLAG (Sigma, F1804), α-rat HA (Roche, 11 867 423 001).
The following secondary antibodies were used in this study: α-mouse IgG HRP-linked whole antibody (GE Healthcare, NA931), α-rabbit IgG HRP-linked whole antibody (GE Healthcare, NA934V), α-rat IgG HRP-linked whole antibody (GE Healthcare, NA935V), Goat α-mouse IgG Alexa Fluor 488 (Thermo Fisher Scientific, A-11001), Goat α-rabbit IgG Alexa Fluor 568 (Thermo Fisher Scientific, A-11011).

200 Morpholino-injected embryos were collected at 48 hpf, and fixed in 1% formaldehyde, then homogenized and sonicated into 300-500 bp chromatin fragments. Chromatin fragments were immunoprecipitated overnight with Anti-RNA polymerase II CTD repeat YSPTSP and S5 antibody (Abcam, ab817 and ab5408, 1 ug per 0.5 ml of sonicated chromatin) or IgG (Santa Cruz, 1ug per 0.5 ml of sonicated chromatin). Chromatin Immunoprecipitation (ChIP) Assay Kit (Beyotime) was used to obtain targeted chromatin fragments. Nucleic acids were purified by DNA purification kit (TIANGEN) and analyzed by Quantitative RT-PCR according to the previous procedure. The sequences of qRT-PCR primers are listed in TableS1.

Western-blot analysis was performed on histones extracts prepared from 1-week-old seedlings as described previously (Xu et al., 2008 (link)). Protein were separated by 15% SDS-PAGE and transferred onto Immobilon-P membranes (Millipore) using a Trans-Blot semi-dry transfer cell (Bio-Rad). Intensity of individual bands was quantified using ImageJ densitometry software (NIH).
Chromatin immunoprecipitation (ChIP) assays were performed according to the previously described method (Liu et al., 2016 (link)). Antibodies used to precipitate chromatin were anti-H3 (05-499; Millipore), anti-trimethyl-H3K4 (07-473; Millipore), anti-trimethyl-H3K36 (ab9050; Abcam) and anti-total RNA polymerase II (RNAPII) CTD repeat antibody (ab817, Abcam), together with protein A magnetic beads (Magna-ChIP, Millipore). DNA was purified with the NucleoSpin Gel and PCR Clean-up kit (Macherey−Nagel, Düren, Germany) and analyzed by real-time PCR (LightCycler 480II; Roche in conjunction with the SYBR Green Master mix) using gene-specific primers listed in Supplementary Table S1. Data were analyzed as described in Zhao et al., 2019 (link) for H3K4me3 and H3K36me3 and in Yang et al., 2016 (link) for RNAPII. A mock control was done using uncoupled magnetic beads (Supplementary Figure S5).

Cells were lysed in Laemmli sample buffer (Laemmli, 1970 (link)) supplemented with 10% 2-mercaptoethanol (133-1457; Wako) and incubated at 95°C for 5 min to denature proteins. Then, SDS-PAGE was performed to separate the proteins. Proteins in the gel were transferred to an Immobilon-P membrane (IPVH00010; Merck) and blocked with PBS-T containing 5% nonfat milk (190-12865; Wako) for 30 min at room temperature. Subsequently, the proteins were blotted with antibodies at the indicated dilutions: rabbit anti–histone H2B (ab1790; Abcam) at 1:10,000 or 1:20,000; rabbit anti–HaloTag (G9281; Promega) at 1:1,000; mouse anti–CTD of RPB1 (ab817; Abcam) at 1:1,000; mouse anti–α-Tubuline (T6199; Sigma-Aldrich) at 1:5,000; mouse anti–CDK9 (sc-13130; Santa Cruz) at 1:500; horseradish peroxidase-linked goat anti–rabbit IgG whole antibody (170-6515; Bio-Rad) at 1:5,000 for anti-H2B and anti-HaloTag; HRP-linked goat anti–mouse IgG whole antibody (170-6516; Bio-Rad) at 1:5,000 for anti-CTD, anti–α-Tubuline, and anti-CDK9. Signal detection was performed by using the Immobilon Western Chemiluminescent HRP substrates (WBKLS0500; Merck) with a chemiluminescence CCD imaging system EZ-Capture MG (ATTO).

ChIP-seq was conducted as previously described [43 (link)-45 (link)]. The following antibodies were used in each experiment: anti-histone H3K4me3 antibody (Abcam, Cambridge, UK, ab1012) ,anti-RNA polymerase II (Abcam ab817), monoclonal anti-H3K27me3 antibody (Abcam, ab6002), polyclonal anti-H3K27Ac antibody (Abcam, ab4729), polyclonal anti-H3K36me3 antibody (Abcam, ab9050) For ChIP-seq, Illumina’s Eland was used to map the 36 bp reads to the reference human genome (hg19). Peaks were called by MACS 1.4.1 [24 (link)] at default settings.