Predicted open reading frames for each transcription factor were amplified from cDNA generated using RNA to cDNA EcoDry premix (Clontech). Amplified transcription factor sequences were inserted into an expression vector containing T7 and SP6 promoters upstream of HALO tag as previously described (30 (link)). In vitro transcription and translation of transcription factors was performed using Promega TnT T7 Rabbit Reticulocyte Quick Coupled Transcription/Translation System by incubating 1 μg of plasmid DNA with 60 μL of TnT Master Mix and 1.5 μL of 1 mM methionine overnight at room temperature. Expression was verified using Western blot analysis with Promega Anti-HaloTag monoclonal antibody. Single DAP-seq libraries were generated once for each transcription factor, tested, and sequenced once with Illumina MiSeq 2 × 150-bp runs.
Filtered reads were aligned to the N. crassa OR74A genome (v12) using Bowtie2 v2.3.2 (70 (link)). Peak calling was performed using MACS2 v2.1.1 (74 (link)) with P value cutoff at 0.001 and utilizing negative control library alignments. Peaks within 3,000 bp upstream of translation start sites were selected for and annotated using a custom Python script. The same Python script was used for reanalysis of ChIP-seq peaks dataset from Craig et al. (10 (link)) for DAP-seq/ChIP-seq comparisons.
Filtered reads were aligned to the N. crassa OR74A genome (v12) using Bowtie2 v2.3.2 (70 (link)). Peak calling was performed using MACS2 v2.1.1 (74 (link)) with P value cutoff at 0.001 and utilizing negative control library alignments. Peaks within 3,000 bp upstream of translation start sites were selected for and annotated using a custom Python script. The same Python script was used for reanalysis of ChIP-seq peaks dataset from Craig et al. (10 (link)) for DAP-seq/ChIP-seq comparisons.