Hiseq 1500 machine

Genomic DNA was isolated from blood specimens using the Chemagen DNA kit (PerkinElmer, Shelton, CT) and quantified on a NanoDrop spectrophotometer (ThermoFisher Scientific, Charlotte, NC). Subsequently, the DNA was fragmented with a Covaris ultrasonicator instrument (Woburn, MA). Gene coding regions, as well as the flanking intronic sequences, were captured using SeqCap target enrichment probes (Roche, Basel, Switzerland) according to the manufacturer's protocol. The libraries were paired‐end sequenced (2 × 125 bp) on a HiSeq 1500 machine (Illumina, San Diego, Ca). A minimum coverage of 30× was calculated. FastQ files were analyzed with the SeqNext software package (JSI Medical, Ettenheim, Germany).

Aortae from the Ldlr^-/-Apobec1^-/- mice treated with nothing (CTL, N = 5), CLO (N = 5), or TIC (N = 5) were excised from the body and cleaned to remove adjacent connective and adipose tissue. We then isolated RNA individually from each aorta as described previously using RNA purification columns (Qiagen, Germantown, MD) [35 (link)]. We examined the quality of the RNA using the Agilent 2100 Bioanalyzer (Santa Clara, CA) and found that all samples had RNA integrity numbers higher than 8. We prepared the library using the Illumina (San Diego, CA) TrueSeq mRNA sample preparation kit per the manufacturer’s instructions. RNA sequencing was performed using the Illumina HISeq 1500 machine at the University of Texas Medical Branch NGS Core facility. The sequencing run yielded about 15 million reads on average. We aligned all reads using Tophat with the mm10 build of the mouse genome reference and annotation from the University of Southern California downloaded from Illumina’s iGenome website. We then examined transcript assembly and differential expression using Cufflinks with Refseq mRNAs [36 (link)]. Finally, we analyzed the RNA-seq data using the cummerbund package in R [37 (link)].

For gene expression analyses of sorted cell subsets, RNAseq libraries were generated as follows: messenger (m)RNA was reverse transcribed and pre-amplified using the SMART-Seq v4 ultra low input RNA kit (Takara Clontech), followed by library generation using a Nextera XT DNA library prep kit (Illumina). Sequencing of the libraries was done on an Illumina HiSeq 1500 machine.

E. coli HB101, AZ121 and GS035 strains were grown in LB media containing no antibiotics, ampicillin (100 µg/ml) or chloramphenicol (25 µg/ml) respectively (OD595 = 0.6–0.8). Cultures were centrifuged and the re-suspended pellets were seeded onto NGM plates. For one generation feeding experiments, animals were placed on the seeded NGM plates and allowed to grow until young adult stage. For three generation feeding experiments, L1 larval stage animals were placed on the seeded NGM plates and allowed to grow for three generations. Total RNA from harvested animals was isolated using the TriSure reagent (Bioline). Small RNA sequencing libraries of C. elegans were prepared using the NEBNext Multiplex Small RNA Library Prep set (NEB) and the small RNA sequencing libraries of E. coli are prepared using the TruSeq Small RNA set (Illumina). Libraries were either sequenced on a MiSeq or a HiSeq 1500 machine (Illumina). Data analysis was performed as previously described21 (link). Small RNA-seq data is available through NCBI-GEO with accession number GSE64990. qRT-PCR is performed with primers described in Liu et al17 (link). OxyS RNA structure is determined using the Mfold web server22 (link).