Hp_8Gy_7d_2_1_Human_Hepatocyte
RNA-seq Data Processing  Raw reads from the RNA-seq data were first filtered to remove reads with >10% uncertain nucleotides and/or >50% proportion of low-quality (i.e., <20) bases. Reads with adapter contamination were also removed. After quality filtering, hierarchical indexing for spliced alignment of transcripts (HISAT2) was used for mapping reads to the human reference genome 52. Featurecounts, a highly efficient read summarization method, was then implemented to assign reads to both protein coding and long non-coding genomic features 53. SMARTer Stranded V2 RNA sample quality was assessed by BioAnalyzer High Sensitivity RNA Assay (Agilent Technologies Inc., California, USA) and quantified by AccuBlue Broad Range RNA Quantitation assay (Biotium, California, USA). Library construction is performed based on manufacturers recommendation for SMARTer Stranded Total RNA-Seq Kit v2  Pico Input RNA Kit (Takara Bio USA Inc., California, USA). Final library quantity was measured by KAPA SYBR FAST qPCR and library quality evaluated by TapeStation HSD1000 ScreenTape (Agilent Technologies, CA, USA). Average final library size is 450bp and Illumina 8-nt dual-indices were used. Equimolar pooling of libraries was performed based on QC values and sequenced on an Illumina NovaSeq 6000 (Illumina, California, USA) with a read length configuration of 150 PE for 40 M PE reads per sample (20M in each direction). RNA-seq Data Processing  Read quality and adapter contamination were assessed using the FastQC (https://github.com/s-andrews/FastQC, v0.11.9) and low-quality reads were removed using the read trimming tool Trimmomatic (v0.39) 54. After read filtering, the STAR (v2.7.10a) 55aligner was used for mapping reads to the human reference genome (primary genome assembly GRCh38) and assigning read counts to mRNA, miRNA, and lncRNA genomic features. RNA-sequencing Data Analysis Differential gene expression analyses were performed on raw read counts. All pair-wise comparisons for differential gene expression analyses were conducted using the DESeq2 R package 56, and included comparisons between radiation treatment dose levels (4 Gy, and 8 Gy) and control (0 Gy) across three dosage timepoints (6h, 24h, 7d). Genes with an adjusted p-value (FDR) < 0.05 were considered differentially expressed and utilized in downstream analysis. Where noted, more stringent log fold-change thresholds were employed in addition to adjusted p-value filtering.