Dnase 1 treatment step

Aortas were perfused with PBS and then with RNAlater (Qiagen), and the aortic arch (third rib to aortic root) was removed (to get RNA from the most atherosclerotic part of the aorta) and homogenized with FastPrep (Qbiogene). Total RNA was isolated with an RNeasy Mini-kit with a DNAse I treatment step (Qiagen). RNA quality was assessed with a Bioanalyzer 2100 (Agilent Technologies), and RNA quantity with NanoDrop (Thermo Scientific). Global mRNA expression profiles were generated with Mouse Gene 1.0 ST arrays (Affymetrix) according to the manufacturer's protocol. In brief, amplified and biotinylated cRNA was generated from 100 ng of high-quality RNA with the GeneChip WT Sense Target Labeling and Control Reagents kit (No. 900652, Affymetrix). The arrays were hybridized in a GeneChip Hybridization Oven 640, further processed with a Fluidics Station 450, scanned with a GeneArray Scanner 3000 7G, and analyzed with GeneChip Operational Software 2.0. For global gene expression profiling, 18 Ldlr^−/−Apob^100/100Mttp^flox/flox control mice (n = 6, 6, and 6 at week 30, 40, and 50, respectively) and 48 Ldlr^−/−Apob^100/100Mttp^Δ/Δ mice (early lesions: n = 6 immediately after PCL and n = 10 after PCL for 10 weeks; mature lesions: n = 6 immediately after PCL and n = 10 after PCL for 10 weeks; advanced lesions: n = 6 immediately after PCL and n = 10 after PCL for 10 weeks) were used.

We tested cells from three independent experiments using three consecutive passages. Total RNA was extracted using TRizol reagent (15596026, ThermoFisher Scientific) and RNeasy Mini Kit (74104, QIAGEN) with DNase I treatment step (79254, QIAGEN) following the manufacturer’s protocol. The integrity of RNA was determined by RNA ScreenTape (5067-5576, Agilent Technologies) on the Agilent 4200 TapeStation (Agilent Technologies). RNA-seq libraries were prepared starting from 600 ng of total RNA using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina) as recommended by the manufacturer. Half of the oriented cDNA produced from the poly-A+ fraction were PCR amplified (11 cycles). RNA-seq libraries were sequenced on an Illumina HiSeq2500 (Paired-End sequencing 130 × 130 bases, High Throughput Mode). A minimum of 10 million of paired-end reads was produced per library sample. Sequence reads were aligned to the human HG19 reference genome using the Burrows-Wheeler Alignment version 0.6.2.13. Raw and processed data for all samples are available for download from Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE100670. RNA-Seq data were analyzed using a combination of three different tools namely limma [15 (link)], DESeq2 [16 (link)], and edgeR [17 (link)].

Total RNA was extracted using TRIzol reagent (Life Technologies) and RNeasy Mini Kit (QIAGEN) with DNase I treatment step (QIAGEN). cDNA was reversed transcribed from extracted RNA using SuperScript® II Reverse Transcriptase (Life Technologies). Gene expression was measured using SYBR Green Power Mix (Life Technologies) using primers specific for each gene. Additional file 1: Table S4 contains the sequences of all primers used in this study. Relative standard curve method was employed to calculate relative gene expression. Reactions were performed using the Step One Plus Real-Time PCR system (Applied Biosystems). Expression values were taken from mean of triplicates.