ChIP-seq analysis of RNAPII-S2P in Arabidopsis

Chromatin immunoprecipitation (ChIP) was essentially performed as previously described (Antosz et al., 2020 (link); Michl-Holzinger et al., 2022 (link)). Plants (14-DAS in vitro grown) were crosslinked with formaldehyde and used for isolation of nuclei, before chromatin was sheared using a Bioruptor pico device (Diagenode). Immunoprecipitation was performed using antibodies directed against RNAPII-S2P (abcam, ab5095). For ChIP-seq, libraries were generated using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs) and the final libraries (3 replicates each) were sequenced by the Genomics Core Facility at the University of Regensburg using NextSeq 2000 (Illumina). Reads were aligned to the TAIR10 genome (https://www.arabidopsis.org/) using Bowtie2 (Langmead and Salzberg, 2012 (link)) and coverage tracks were calculated with deeptools “bamCoverage”. Downstream analysis was mainly performed using the deepTools2 suite (version 3.5.0) (Ramírez et al., 2016 (link)) and quality control was performed at several steps using FastQC (Ewels et al., 2016 (link)). Reads were mapped against the TAIR10 genome and regions with aberrant coverage or low sequence complexity were removed (Quadrana et al., 2016 (link)). After confirming high pairwise correlations, the biological replicates were merged and CPM normalized.