Anti h3k27me3

ChIP analysis was performed using the EpiTect ChIP OneDay kit (Qiagen). Briefly, infected Calu3 cells were cross-linked, harvested, and frozen at −80°C. Cells were then lysed and chromatin sheared via sonication to generate chromatin fragments between 250 and 1,000 bp. Sonicated samples were then immunoprecipitated with anti-STAT1 (clone C-24; Santa Cruz Biotechnology), anti-H3K4me3 (Qiagen), anti-H3K27me3 (Qiagen), or anti-mouse IgG (Qiagen) as a control. To determine the histone modification distribution, quantitative real-time PCR was performed targeting the 5′UTR (−750 bp—TSS [region 750 base pairs upstream of the transcriptional start site]) of select genes; target and primer information is included in the supplemental material. ChIP results were reported as fold difference (or differential occupancy), allowing comparison across multiple samples. For this, each sample was normalized to the input and then the fold enrichment was calculated using the ΔΔC_T method. The fold difference for each gene was determined by dividing the appropriate time-matched mock by the experimental group. Finally, the fold difference values were converted to log₂ and plotted. Data presented are the means ± standard errors for triplicate samples.

ChIP analysis was performed by using the EpiTect ChIP OneDay Kit (Qiagen). Briefly, infected Calu3 cells were cross-linked, harvested, and frozen at −80 °C. Cells were then lysed and chromatin sheared via sonication to generate chromatin fragments between 250 and 1,000 base pairs. Sonicated samples were then immunoprecipitated with anti-STAT1 (clone C-24; Santa Cruz Biotechnology), anti-H3K4me3 (Qiagen), anti-H3K27me3 (Qiagen), or anti-mouse IgG (Qiagen) as a control. To determine the histone modification distribution, quantitative real-time PCR (qPCR) was performed by targeting the 5′ UTR (−750 bp to transcription start site) of select genes; target and primer information are included in Supporting Information. ChIP results were reported as fold difference (or differential occupancy), allowing comparison across multiple samples. For this, each sample was normalized to the input, and then the fold enrichment was calculated by using the ΔΔCt method. The fold difference for each gene was determined by dividing the appropriate time-matched mock by the experimental group. Finally, the fold difference values were converted to log2 and plotted. Data presented are the means ± SEs for triplicate samples.

ChIP analysis for NGS sequencing was performed by using the MAGnify ChIP System (Invitrogen). Briefly, for ChIP analysis, Calu3 cells were plated (∼1.5 × 10⁶ per well) and infected with H5N1-VN1203 or H1N1-09 at an MOI of 5 and harvested at 0 and 12 hpi; cultures inoculated with PBS alone served as time-matched mock controls. Sonication conditions were chosen to result in the desired size distribution of the sonicated chromatin between 250 and 1,000 bp. Sonicated samples were then immunoprecipitated with anti-H3K4me3 (Qiagen) and anti-H3K27me3 (Qiagen). To determine genome-wide histone modification, ChIP DNA was subjected to next-generation sequencing (NGS) on a TruSeq ChIP Library Preparation Kit (Illumina) and sequenced on an Illumina HiSeq. NGS data analysis was performed by utilizing the CLC Genomics Workbench. The Histone ChIP-Seq plugin provided analysis tools for a complete histone modification analysis. Briefly, paired end reads were mapped against the human GRCh37/hg19 reference genome by using a stringent alignment setting (mismatch cost = 2). Peaks were called against the time-matched mock reference reads. To determine specific genomic regions of histone modification enrichment, the maximum P value for peak calling was set to P < 0.05 to detect regions that had a significant fit with the peak shape.