The quality of the sequenced reads was assessed with FastQC and QoRTs. Unless stated otherwise, plots involving high- throughput sequencing data were created using R and ggplot2.
DNA sequencing reads were aligned with default parameters to the mouse reference genome (GRCm38.p6) using STAR. Gene expression estimates were obtained with featureCount using composite gene models (union of the exons of all transcript isoforms per gene) from Gencode (version M17).
DEGs were determined using DESeq2 with Wald tests with a q-value cutoff of 0.05 (Benjamini–Hochberg correction).
Heatmaps were created using DESeq2 normalized read counts after variance stabilizing transformation of genes identified as differentially expressed by DESeq2. Rows were centered and scaled.
Gene set enrichment analyses were done using fgsea with the fgseaMultilevel function. Genes were ranked based on the DESeq2 Wald statistic. Gene sets with an FDR < 0.05 were considered enriched. Gene ontology analysis was performed on up- and down-regulated DEGs using the clusterProfiler R package. Only GO categories enriched using a 0.05 false discovery rate cutoff were considered.
Reads were aligned to the mouse reference genome (version GRCm38) with BWA-backtrack v0.7.17. Post-alignment filtering was done with samtools and Picard tools to remove unmapped reads, improperly paired reads, nonunique reads, and duplicates. Peaks were called with MACS2, ,and peaks with adjusted P values smaller than 0.01 were excluded. Consensus peak sets were generated for each condition if a peak was found in at least two replicates. Reproducible peaks from each condition were merged with DiffBind to create an atlas of accessible peaks, which was used for downstream analyses. The peak atlas was annotated using the ChIPseeker and TxDb.Mmusculus.UCSC.mm10.knownGene. Blacklisted regions were excluded (https://sites.google.com/site/anshulkundaje/projects/blacklists).
Regions where the chromatin accessibility changed between different conditions were identified with DESeq2, and only Benjamini–Hochberg corrected P values < 0.05 were considered statistically significant.
Genome coverage files were normalized for differences in sequencing depth (RPGC normalization) with bamCoverage from deepTools. Replicates were averaged together using UCSC-tools bigWigMerge. Merged coverage files were used for display in Integrated Genomics Viewer.
Heatmaps based on the differentially accessible peaks identified between TCROT1(-CD4) and TCROT1(+CD4) were created using profileplyr and ComplexHeatmap, by binning the region +/− 1kb around the peak summits in 20bp bins. To improve visibility, bins with read counts greater than the 75th percentile + 1.5*IQR were capped at that value.
For identifying motifs enriched in differentially accessible peaks, we utilized HOMER via marge. HOMER was run separately on hyper- or hypo-accessible peaks with the flags -size given and -mask. Motifs enriched in hyper- or hypo-accessible peaks were determined by comparing the rank differences (based on P value). The consensus peakset identified by DiffBind was used as the background set.
| Software | Version | Authors | URL |
|---|---|---|---|
| STAR | v2.6.0c | Dobin et al. | GitHub |
| featureCounts | v1.6.2 | Liao et al. | Subread |
| R | v4.1.0 | R Core Team | CRAN |
| DESeq2 | v1.34.0 | Love et al. | Bioconductor |
| ggplot2 | v3.4.1 | Wickham | CRAN |
| pheatmap | v1.0.12 | Kolde | CRAN |
| fgsea | v1.20.0 | Korotkevich et al. | Bioconductor |
| clusterProfiler | v4.2.2 | Guangchuang et al. | Bioconductor |
| BWA | v0.7.17 | Li et al. | Bio-BWA |
| samtools | v1.8 | Danecek et al. | HTSlib |
| Picard tools | v2.18.9 | Broad Institute | GitHub |
| MACS | v2.1.1 | Zhang et al. | GitHub |
| DiffBind | v3.4.11 | Stark and Brown | Bioconductor |
| ChIPseeker | v1.30.3 | Wang et al. | Bioconductor |
| deepTools | v3.1.0 | Ramírez et al. | Documentation |
| bigWigMerge | UCSC KentUtils | Kuhn et al. | GitHub |
| IGV | - | Robinson et al. | Documentation |
| profileplyr | v1.10.2 | Carroll and Barrows | Bioconductor |
| ComplexHeatmap | v2.15.1 | Gu | Bioconductor |
| HOMER | v4.10-0 | Heinz et al. | HOMER |
| marge | v0.0.4 | Amezquita | GitHub |