Hiseq 3000 4000

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).

ATAC-seq was performed as previously described^{15 (link), 38 (link)}, with minor adaptations. In each experiment, a maximum of 50,000 sort-purified cells were collected at 300 g for 5 min at 4 °C. After centrifugation, the pellet was carefully resuspended in the transposase reaction mix (12.5 μl 2xTD buffer, 2 μl TDE1 (Illumina), 10.25 μl nuclease-free water, and 0.25 μl 1% digitonin (Promega)) for 30 min at 37 °C. Following DNA purification with the MinElute kit eluting in 11 μl, 1 μl of eluted DNA was used in a quantitative PCR (qPCR) reaction to estimate the optimum number of amplification cycles. The remaining 10 μl of each library were amplified for the number of cycles corresponding to the C_q value (i.e., the cycle number at which fluorescence has increased above background levels) from the qPCR. Library amplification was followed by SPRI (Beckman Coulter) size selection to exclude fragments larger than 1,200 bp. DNA concentration was measured with a Qubit fluorometer (Life Technologies). Library amplification was performed using custom Nextera primers^{15 (link)}. Libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq 3000/4000 platform and the 50-bp single-end configuration. Chromatin accessibility mapping by ATAC-seq was done in two biologically independent experiments. Sequencing statistics are provided in Supplementary Table 1.