ChIP-seq and RNA-seq of RAW Cells

For ChIP-seq, ∼10 × 10⁶ of untreated or stimulated (1 or 4 h after LPS) RAW cells were used for each experiment. Cells were crosslinked with 0.75% formaldehyde for 10 min at room temperature, and chromatin was sonicated as described previously (29 (link)). Each chromatin input was immunoprecipitated with 10 μg of the following antibodies: H3K4me1 (Abcam 8895), H3K4me3 (Active Motif 39159), H3K36me2 (Abcam 9049), H3K79me2 (Abcam ab3594), H3K36me3 (Abcam 9050), H3K27ac (Abcam 4729), H3K27me3 (Cell Signalling 9733) and Pu.1 (Santa Cruz sc-352). After immunoprecipitation, beads were washed three times in buffer A (20 mM Tris–HCl [pH 7.6], 2 mM EDTA, 0.1% SDS, 1% Triton-100 and 150 mM NaCl), once in buffer B (20 mM Tris–HCl [pH 7.6], 2 mM EDTA, 0.1% SDS, 1% Triton-100 and 300 mM NaCl) and once in TE containing 50 mM NaCl. DNA was eluted in TE containing 2% SDS and de-crosslinked overnight at 65°C. DNA was purified by QIAquick columns (QIAGEN) and quantified with Picogreen. Sequencing libraries were generated as previously described (53 (link),54 (link)) and sequenced on an Illumina HiSeq2000. Total RNA was extracted from 2 × 10⁶ cells using RNeasy Kit (QIAGEN) with DNase I treatment. Libraries were then prepared using TruSeq RNA sample preparation Kit (Illumina) after depleting ribosomal RNA and sequenced on an Illumina HiSeq2000.