Truseq mrna sample preparation kit

To profile baseline transcriptomic differences, total RNA was isolated from three control fibroblast lines and three SSc fibroblast lines (derived from lesional skin) using Trizol and RNeasy isolation columns (Qiagen) per manufacturer’s protocol. DNA was digested with on-column DNase (Qiagen) treatment. The quality of the RNA used for sequencing was determined using an Agilent 2100 Bioanalyzer; all samples had RNA integrin numbers (RIN) of at least 9.60. mRNA was enriched by poly-A selection, prepped using an Illumina TruSeq mRNA sample preparation kit, and sequenced by Illumina HiSeq 2000. Adapter sequences from one-hundred base-pair paired-end FASTQ reads were trimmed using Trimgalore (https://github.com/FelixKrueger/TrimGalore). Trimmed sequence reads were aligned to hg19 using RSEM (35 (link)). Differential gene expression analysis was performed using DESeq2 package (36 (link)) with the gene count output from RSEM. The top differentially expressed genes (false discovery rate [FDR] < 0.05) were used to generate heatmaps and for subsequent gene ontology (GO) analyses. To assess the therapeutic efficacy of JQ1, total RNA was isolated from three control fibroblast lines and three SSc fibroblast lines using Trizol after 48 hours treatment with DMSO or JQ1. Samples were processed for RNA-seq as detailed above.

RNA was purified from tissue chunks of 3 primary tumors, 7 lung metastases, 8 normal lung and 12 normal mammary gland tissues (Supplementary Table 1) following the manufacturer’s protocol (PicoPure RNA Isolation Kit, ThermoFisher Scientific). Gene expression profiles were generated by mRNA-sequencing using an Illumina HiSeq 2000/2500. Briefly, mRNA libraries were made from total RNA using the Illumina TruSeq mRNA sample preparation kit and sequenced on an Illumina HiSeq 2000/2500 using a 2×50bp configuration with an average of 136 million read pairs per sample. Quality-control-passed reads were aligned to the mouse reference genome (GRCm38) using STAR^{80 (link), 81 (link)}. The alignment profile was determined by Picard Tools v1.64 (http://broadinstitute.github.io/picard/). Aligned reads were sorted and indexed using SAMtools and translated to transcriptome coordinates then filtered for indels, large inserts, and zero mapping quality using UBU v1.0 (https://github.com/mozack/ubu). Transcript abundance estimates for each sample were performed using SALMON^{82 (link)}. Raw SALMON read counts for all RNAseq samples were normalized to a fixed upper quartile.