DNA-RNA immunoprecipitation in WT, CDK8-KO, and CDK8/HDAC3-KO HAP1 cells treated with ATRi or DMSO

DRIP assays were performed as previously described, in three biological replicates with the following amendments (during analysis we used 2 replicates of CDK8/HDAC3 dKO cells in both DMSO and ATRi treated samples, as we omitted a low-quality replicate for each). HAP1 cells were seeded on 10-cm plates for 16 h followed by a 6 h treatment with 1 µM AZD6738. Cells were washed once with ice cold PBS, lysed in cytoplasmic lysis buffer for 10 min on ice followed by a single wash with cytoplasmic lysis buffer. Nuclei were lysed in nuclei lysis buffer and the lysate incubated for 1 h at 55?C with 300 rpm agitation. To precipitate the DNA, 3 M sodium acetate pH 5.2 was added (300 mM final) followed by transfer into 2.5X volume of 100% ethanol. The DNA was then washed in 85% ethanol, air dried and re-suspended in 1X TE buffer. 50 µg of DNA was sonicated using a Diagenode Bioruptor on high settings for 13 cycles of 10 sec on/30 sec off and sonication confirmed by running the samples on a 1.5% agarose gel. All samples were equilibrated into 1X DRIP buffer and 20 µg was immunoprecipitated overnight at 4?C with 5 µg S9.6 antibody pre-conjugated to 100 µL Dynabeads protein G. The beads were washed once in 1X DRIP buffer, once in 1X DRIP with 500 mM NaCl, once in LiCl buffer, and twice in 1X TE. DNA was eluted by incubation with 1X TE pre-added to 0.5% SDS and 0.5 mg/mL proteinase K for 30 min at 55?C. Eluted DNA was purified using phenol:chloroform:isoamylalcohol and following this, precipitated by ethanol. DNA was resuspended in 65 µL nuclease free water. 5 ng of DRIP DNA from each of three biological replicates from each condition were used for library preparation using the Thruplex DNA-seq kit for Illumina (TAKARA, R400674) according to the manufacturer's protocol, using 12 PCR cycles for IP samples and 8 PCR cycles for input samples. Libraries were then sequenced as 2 lanes of S1 PE50 on the Illumina NovaSeq6000.