ChIP assays were performed as previously described (Fang et al., 2015), using antibodies against AGO1 (Qi et al., 2005), myc (Abcam, ab32), Pol II (Abcam, ab817), Pol II Ser2P (Abcam, ab5095), or Pol II Ser5P (Abcam, ab5131). For GRO-seq, ~5X10^6 nuclei were run on for 5 min at 30 °C. The reactions were stopped by addition of Trizol and RNAs were extracted. After digestion with Terminator exonuclease (Epicentre) and fragmentation, nascent RNAs were enriched twice for 5-bromo-UTP (BrUTP) by immunoprecipitation. For RNA-seq, total RNA was purified from 10-day-old seedlings using RNeasy Plant Mini Kit (QIAGEN). For sRNA-seq, nuclei were isolated from 10-day-old Col-0 and coi1-1 seedlings without or with MeJA treatment as described (Wang et al., 2011b). Nuclear lysates were used for immunopurification of AGO1 complexes as described (Mi et al., 2008). To characterize nuclear AGO1-associated sRNAs, sRNAs were extracted from the immunoprecipitates as described (Mi et al., 2008). Libraries for ChIP-seq were generated using the NEXTflex ChIP-seq Kit (Bioo Scientific) according to the manufacturer's instructions; Libraries for GRO-seq were performed as described (Wang et al., 2011a); Libraries for RNA-seq were generated using NEXTflex RNA-Seq Kit (Bioo Scientific) or NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB) according to the manufacturer's instructions; Libraries for sRNA-seq were generated using NEBNext Small RNA Library Prep Set for Illumina (NEB).
GEO Accession
GSM2522240