Hiseq 3000 system

Analyses were conducted at the Yerkes NHP Genomics Core. Liver mRNA was collected and extracted from PAXgene tubes using on-column DNase digestion and assessed for integrity and quantity using an Agilent Bioanalyzer (Agilent Technologies) and a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Libraries were prepared using the Illumina TruSeq mRNA stranded kit. Briefly, 500–1,000 ng of globin-depleted RNA was used for library preparation. ERCC synthetic spike-in controls 1 or 2 (Ambion) were added to each total RNA sample and processed in parallel. Amplified libraries were validated using the Agilent 4200 TapeStation and quantified using a Qubit fluorometer. Libraries were normalized and pooled, followed by clustering on a HiSeq 3000/4000 flowcell using the Illumina cBot. The clustered flowcell was then sequenced on the Illumina HiSeq 3000 system employing a single-end 101-cycle run, with multiplexing to achieve approximately 20 million reads per sample. FasQ files were uploaded to the BioJupies^{53 (link)} RNAseq cloud pipeline where differential expression analysis and downstream pathway and transcription factor enrichment analyses were performed. RNA-sequencing data are publicly available at the Gene Expression Omnibus: GSE145012 (GF/CV comparison) and GSE185525 (VB treatment to GF mice).

After quality check with samples reaching RNA integrity values RIN > 8, up to 1 µg of total RNA was used for poly-A enrichment and subsequent library preparation with the TrueSeq stranded mRNA Kit from Illumina (HUES8 n=6). Subsequent RNA-seq was performed on a HiSeq 3000 system (Illumina, single read, 1×50bp) at the Biomedical Sequencing Facility (BSF) of the CeMM in Vienna, Austria.
Reads were aligned with STAR aligner (Version 2.5.2b) on human genome hg38 and using Ensembl Transcriptome Annotation e87 as transcriptome reference. STAR parameters followed options suggested by the ENCODE project. Next, DESeq2 (version 1.22.1) was used for normalization and differential expression analysis. Only genes with at least 10 reads, an adjusted p-value < 0.05 and abs(FC) > 1.5 were considered. Clustering of gene expression is based on a fuzzy c-means algorithm (R package e1071). This pipeline was performed to cluster data from HUES8 (ESC, DE, PE and PP stages on WT, ONECUT1 KO and truncated ONECUT1 cells).

Sonicated and size-selected DNAs (immunoprecipitated DNA that was untreated or treated with etoposide or RNaseH, and input DNA) were processed according to the Illumina Genome DNA library preparation protocol, and sequenced with a HiSeq 2000 or a HiSeq 3000 system with 50 bp single-read sequencing protocol. On average, 30–40 million reads were generated for each DNA sample, and then aligned to the UCSC hg19 genome build using BWA. Peak calling was performed using the MACS2^{40 (link)} algorithm. Similar to ref. 17, we used a peak calling parameter with fivefold up to 30-fold enrichment over corresponding input DNA as control (MACS2 parameters: -g 2.7e9 -q 0.05 -B -m 5 30).