ATACseq differential accessible (DA) peak analyses

Raw reads were preprocessed by Trimmomatic to remove adapters and low-quality reads [113] (LEADING:20 TRAILING:20 SLIDINGWINDOW:5:25 MINLEN:36) before being aligned to schMedS2 ({"type":"entrez-nucleotide","attrs":{"text":"NNSW00000000.1","term_id":"1267084223","term_text":"NNSW00000000.1"}}NNSW00000000.1) planarian genome assembly [54]. The genome was indexed using bowtie2-build and fastq files were aligned to the genome using bowtie2 [118]. Reads mapped to the nonmitochondrial genome and with mapping quality scores > 30 were kept for downstream analysis using SAMtools (v 1.16.1) [119, 120] (samtools view -b -q 30). SAMtools was used to determine insert sizes—a successful ATACseq experiment should periodic fragment sizes correlating with nucleosome size. One library failed (control_1) and thus was excluded from further analysis (Additional file 13). Initial peak calling of each library were conducted by MACS2 (version 2.7.9) (--nolambda --nomodel --cutoff-analysis -B --keepdup all --gsize 7.6e8) [121]. Peaks showing enriched signals in one condition versus the other were identified using DiffBind package (version 3.0.15) [122] (DESeq2, FDR < 0.05, no fold cut-off). We used the output from DiffBind for all downstream analyses. The R package ChIPSeeker (version 1.26.2) [123, 124] was used to annotate peaks to the closest mapped transcript and generate pie chartdeepTools (version 3.5.0) [125] was used to generate normalized coverage files (bamCoverage –binSize 1 –normalizeUsing CPM) and WiggleTools [126] was used to generate average bigwig files for replicates of the same condition displayed in the genome browser tracks. IGV was used to display bigwig files (peak data) against genome tracks [127]. GMAP [128] was used to generate a gff file mapping the planarian Smed_ASXL transcriptome (NCBI Bio Project PRJNA215411) to the genome, which was displayed with SMESG high-confidence annotations [129]. Peak files were group-autoscaled for visualization purposes on IGV [127].