Ratio and Key Clinicodemographic Data Table

Blood samples were obtained from the three large urology practices. Blood samples were collected in four K2EDTA BD VacutainerTM tubes (Cat. No. 366643, BD Biosciences, San Jose, California) and transferred to the processing locations on ice at 4˚C and processed 4 hours after draw time.Blood was pooled from 3 blood tubes at 4˚C and split into 1/3 and 2/3 aliquots for CD2 and CD14 cell type isolations, respectively. Specially formulated positive selection magnetic-activated cell sorting (MACS) microbeads using anti-CD2 antibodies and anti-CD14 antibodies (Cat. No. 130-101-329 and 130-101-328, respectively, Miltenyi Biotech, Bergisch Gladbach, Germany) were added to the aliquots of blood at a volume of 25 µl CD2 beads per 1ml blood and 50 µl CD14 beads per 1 ml blood. Beads were incubated with the blood samples for 10 minutes at 4˚C. The blood-bead suspensions were then processed at 4˚C using a positive selection template on the autoMACS Pro Separator (Miltenyi Biotech) to isolate the CD2 and CD14 cells. Small aliquots of the isolated CD2 and CD14 cells were removed for flow cytometry analysis while the remaining cells were pelleted by a 10-minute centrifugation at 300 x g at 4˚C. Following centrifugation, the supernatant was removed and 700 µL of room temperature QIAzol Lysis Reagent (Cat. No. 79306, Qiagen, Hilden, Germany) was added to each cell pellet and the cell suspension pipetted up and down for 2 minutes to lyse the cells. The suspension was then vortexed for 1 minute to further homogenize the cell lysates and frozen at -80˚C.
RNA extraction was accomplished using the miRNeasy Mini Kit (Cat. No. 217004, Qiagen). In essence, the frozen CD2 and CD14 cell samples (-80˚C) were thawed in a 37˚C dry bath (~2.5 minutes) and incubated at room temperature for 5 minutes prior to the addition of 140 µL of chloroform and shaken vigorously for 15 seconds. Following a 3 min room temperature incubation, the samples were centrifuged at 12,000 x g (4˚C, 15 min). The upper clear aqueous phase (~350 µL) was transferred to a 2 mL collection tube that was then placed inside the QIAcube (Cat. No. 9001292, Qiagen), and poly(A) RNA was extracted using the miRNeasy Mini Kit per manufacturer’s protocol. The quality and quantity of each RNA sample was determined on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, California).
mRNA was purified from approximately 200 ng of total RNA with oligo-dT beads and sheared by incubation at 94˚C. Following first-strand synthesis with random primers, second strand synthesis was performed with dUTP for generating strand-specific sequencing libraries. The cDNA library was then end-repaired, A-tailed, the adapters were ligated, and second-strand digestion was performed by uracil-DNA-glycosylase.
Sample concentrations were normalized to 10 nM and loaded onto Illumina Rapid or High-output flow cells at a concentration that yields 130-250 million passing filter clusters per lane. Samples were sequenced using 75 bp paired-end sequencing on an Illumina HiSeq 2500 according to Illumina’s protocols. The 6 bp index is read during an additional sequencing read that automatically follows the completion of read 1.
Signal intensities were converted to individual base calls during using the system's Real Time Analysis (RTA) software. Sample demultiplexing was performed using Illumina's CASAVA 1.8.2 software suite. Demultiplexed raw (FASTQ) RNA sequencing data was processed using Trimmomatic for adaptor trimming, Bowtie2 for alignment to the UCSC (University of California, Santa Cruz) hg19 transcriptome, and Express for quantification. Processed reads yielded counts for 23,368 transcripts (gene symbols), corresponding to 29.8±7.5 million and 33.9±7.5 million mapped reads for CD2 and CD14 samples, respectively. Sample normalization to account for RNA concentration differences was performed using Trimmed Mean M-Value (TMM) normalization.
Initial filtering of the transcriptomic data resulted in a reduced set of 18,703 transcripts with observed expression (nonzero counts) in at least 15% of the samples in either CD2 or CD14. The CD2-normalized CD14 signal was used as model input. The normalization consists of subtracting the log-transformed CD2 counts from the log-transformed CD14 counts per patient, on a gene-by-gene basis. This is also referred to as the log(CD14/CD2) ratio.