Thruplex kit

Generation of RNA sequencing (RNA-seq) libraries was with the Ovation SoLo RNA-seq System for Drosophila (NUGEN, 0502-96). All reactions included integrated Heat-Labile Double-Strand Specific DNase treatment (ArcticZymes, catalog no. 70800-201). The DNA-sequencing libraries for TaDa were generated using the Takara ThruPLEX Kit (catalog no. 022818). For rat RNA-seq, libraries were prepared using the Nugen Ovation Model organism (Rat #0349-32) with 1/10th ERCC spike-in mix. These libraries were run on a NextSeq instrument using a HO 150 cycle cit (75 × 75 bp paired-end reads). All Drosophila libraries were sequenced on the Illumina NextSeq platform (High-output kit v2 75 cycles) at the University of Michigan Genomics Core facility.

Chromatin Immunoprecipitation and library synthesis were performed as described [45 (link)]. Briefly, 10⁷ cells were cross-linked with DMEM media with 1% formaldehyde for 15 min and quenched with 2.5 M glycine. After lysis, nuclei were sonicated with a Bioruptor probe (Diagenode) in an ice water bath set to medium strength in increments of 10 s on and 10 s off for 10 min. For the immunoprecipitation (IP), 5 μg of H3K27ac (Abcam ab4729) or 10 μg of H3K4me3 (Abcam ab8580) antibody was used for each replicate. Library synthesis was performed using the ThruPLEX kit (Takara) and quantified using the NEBNext Library Quant Kit (NEB). Sequencing was performed on an Illumina HiSeq2000 sequencer generating an average of 16 million reads per library. Two biological replicates were generated for each cell line. Reads were aligned and processed as described above. Reads for each IP target were merged and a consensus peak set was called using MACS2 with option –broad. For analyses requiring gene transcription start sites (TSS), TSS data in hg19 coordinates were downloaded from BioMart using the R package biomaRt [46 (link), 47 (link)]. For all analyses, only TSS of expressed genes were used. For upset plots comparing the three cell lines, peaks were called within each cell line. Peaks overlapping the respective consensus peak set were then used for plotting.

RNA sequencing (RNA-seq) libraries were generated using the Ovation SoLo RNA-seq System for Drosophila (NUGEN, 0502-96). All reactions included integrated Heat-Labile Double-Strand Specific DNase treatment (ArcticZymes, catalog no. 70800-201). DNA-sequencing libraries were generated using the Takara ThruPLEX Kit (catalog no. 022818) using 3-ng input and 10 cycles of PCR. All libraries were sequenced on the Illumina NextSeq platform (High-output kit v2 75 cycles) at the University of Michigan core facility.