Total sample RNA was extracted using Trizol with Genelute LPA (Sigma) as a carrier, and SMARTer Ultra Low RNA kit for Illumina Sequencing (Clontech) was used for cDNA synthesis according to the manufacturer’s protocol. cDNA was sheared with the Covaris device and processed according to the TruSeq RNA Sample Preparation v2 Guide (Illumina). Amplified sample libraries were subjected to paired-end sequencing (2 × 75 bp) and aligned against mm10 using TopHat.17 (link) Gene expression levels were quantified by the fragments per kilobase of exon per million fragments mapped (FPKM) statistic as calculated by Cufflinks18 (link) in the RefSeq Transcriptome database.19 (link) Read counts were determined with HTSeq-count20 (link) and subsequently used for differential expression analysis in DESeq2,21 (link) with default parameters in the R environment. Multiple testing correction was performed by the Benjamini-Hochberg procedure on the calculated P values to control the false discovery rate (FDR).
For gene set enrichment analysis (GSEA), a ranking metric was defined for each gene as the log10 of the adjusted P value calculated by DESeq2 with the sign of the log2 fold change. The ranked gene list was tested against a customized version of the C2 MSigDB collection, incorporating datasets on HSC quiescence from the literature3 (link),22-24 (link) (supplemental Table 1).
For gene set enrichment analysis (GSEA), a ranking metric was defined for each gene as the log10 of the adjusted P value calculated by DESeq2 with the sign of the log2 fold change. The ranked gene list was tested against a customized version of the C2 MSigDB collection, incorporating datasets on HSC quiescence from the literature3 (link),22-24 (link) (supplemental Table 1).
