Leukolock filter

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless specified otherwise. LeukoLOCK filters and RNAlater were purchased from Life Technologies (Grand Island, NY). The RNeasy Midi kit was obtained from Qiagen, (Valencia, CA). The T7 mouse monoclonal antibody was purchased from Novagen (San Diego, CA). Alexa Fluor 647 goat anti-human IgG and Alex Fluor goat anti-mouse IgG antibodies were purchased from Life Technologies (Grand Island, NY).

Whole blood for RNA was collected in EDTA tubes and filtered using LeukoLOCK filters (Life Technologies). Total RNA was extracted from leukocytes using a modified version of the LeukoLOCK Total RNA Isolation System protocol, and included DNase treatment to remove genomic DNA. RNA quality was assessed using the Agilent 2200 Tapestation, and only samples with RNA Integrity Number (RIN) ≥ 6.0 were used. All libraries were prepared using the Illumina TruSeq mRNA stranded protocol following the manufacturer’s instructions. Samples were sequenced at the McGill University and Genome Quebec Innovation Centre (Montreal, Canada) using the Illumina HiSeq4000 with 100nt paired-end reads. FASTXToolkit and Trimmomatic were respectively used for quality and adapter trimming. Tophat2, using bowtie2 was used to align the cleaned reads to the reference genome. Reads that lost their mates through the cleaning process were aligned independently from the reads that still had pairs. Quantification on each gene’s expression was estimated using HTSeq-count and a reference transcript annotation from ENSEMBL. Counts for the paired and orphaned reads for each sample were added to each other. Normalization was conducted on the resulting gene matrix using DESeq2. All RNA expression values were log2 transformed for data analysis and adjusted for age, gender, and RIN.