Transcriptome Analysis of SHMT2 Knockout

RNA was isolated from cell lines using RNeasy Plus kit (Quiagen) according to the manufacturers´ recommendation. Following the depletion of ribosomal RNA libraries were prepared according to the TruSeq Stranded Total RNA protocol (Illumina) and sequencing was performed on a HiSeq 2500 (Illumina). Analysis was performed using the Galaxy system^{45 (link)} and the R software package⁴⁶ . Adapter sequences were trimmed using Cutadapt (Galaxy version 1.6)⁴⁷ and the trimmed reads were then mapped with TopHat (Galaxy Version 0.9)^{48 (link)} to the GRCh38 reference using ENSEMBL version 80 genes as known splice junctions. The read counts per gene were determined using htseq-count (Galaxy Version 0.6.1galaxy1)^{49 (link)} in ‘union’ mode. Differential expression analysis was performed in R using DESeq2 1.12.3 package^{50 (link)}. SHMT2 knockout gene expression (log2 reads per kilobase of transcript per million mapped reads (RPKM)) was graphed relative to the wild-type and the re-expressed cell lines.