title: RNA-seq of musculus embryonic fibroblasts from CTRP6-KO mice
design: RNA-Seq of 12 samples of CTRP6-KO MEF was performed using NextSeq184 500 Illumina sequencing technology. The experiment was done in two batches (six samples in185 each) and within each experiment 3 biological replicates of the treatment group (rCTRP6) and 3186 biological replicates of the control (con). Initial analysis of the raw sequence reads was carried187 out using the NeatSeq-Flow platform. The sequences were quality trimmed and filtered using188 Trim Galore (v0.4.5) and cutadapt (v1.15). Alignment of the reads to the mouse genome189 (GRCm38) was done using STAR (v2.5.2a). The number of reads per gene per sample was190 counted using RSEM (v1.2.28). Quality assessment (QA) of the process was carried out using191 FASTQC (v0.11.8) and MultiQC (v1.0.dev0). After trimming, each sample had on average of 79M192 reads with an average sequence length of 74.4bp. Statistical testing for identification of193 differentially expressed genes, batch correction, gene annotation, clustering and enrichment194 analysis were performed with the DESeq2 module within the NeatSeq-Flow platform. Batch195 correction was done using the SVA/Combat R package. Gene annotation was done using the196 "AnnotationHub" R package (snapshot: 2020-04-27). After examining the PCA of the normalized197 count it was clear that one of the samples was very different relative to all other samples and198 therefore was removed from downstream analysis. The statistical analysis was done using the199 DESeq2 [5] R package. For comparison (Contrast) between treatment group (rCTRP6) and200 control (con), the statistical model considered two effects: the treatment group and the batch201 (Experiment). The analysis produced p-value, FDR-adjusted p-value and fold of change (FC) per202 gene. Genes with FDR adjusted p-value < 0.05 were considered Differentially Expressed (DE).203 KEGG Enrichment analysis was done using the clusterProfiler (v3.16.0) R package.