For all RNA-seq experiments, raw reads were processed with Trim Galore!
(version 0.4.1) (Babraham Bioinformatics;https://www.bioinformatics.babraham.ac.uk/projects/trim_galore )
to remove adapter sequences and aligned with STAR (version 2.4.2)48 (link) to the human genome (GRCh38,
primary assembly) using the GENCODE annotation (version 23). Differential
expression analysis was performed in R (version 3.2.1) (http://www.R-project.org/ ) using the DESeq2
package (version 1.10.0)49 (link).
Gene set enrichment analysis was performed with GSEA2 (version 2.2.0)
and gene sets from MSigDB (version 5.0)50 (link),51 (link). We used the
“preranked” algorithm to analyze gene lists ranked by the negative
decadic logarithm of adjusted p-values obtained from the differential-expression
analysis with DESeq2. To separate up- and down-regulated genes, we artificially
assigned a negative sign to values for downregulated genes (thus using the
decadic logarithm of adjusted p-values). We used the options –nperm 1000,
-set_max 1500, and –set_min 5.
(version 0.4.1) (Babraham Bioinformatics;
to remove adapter sequences and aligned with STAR (version 2.4.2)48 (link) to the human genome (GRCh38,
primary assembly) using the GENCODE annotation (version 23). Differential
expression analysis was performed in R (version 3.2.1) (
package (version 1.10.0)49 (link).
Gene set enrichment analysis was performed with GSEA2 (version 2.2.0)
and gene sets from MSigDB (version 5.0)50 (link),51 (link). We used the
“preranked” algorithm to analyze gene lists ranked by the negative
decadic logarithm of adjusted p-values obtained from the differential-expression
analysis with DESeq2. To separate up- and down-regulated genes, we artificially
assigned a negative sign to values for downregulated genes (thus using the
decadic logarithm of adjusted p-values). We used the options –nperm 1000,
-set_max 1500, and –set_min 5.
