Nucleic acids were extracted from tonsil specimens (Supplementary Fig. 3b ) and PBMCs (patients 1 to 3 in Supplementary Fig. 4c ) using AllPrep DNA/RNA Mini kits (Qiagen). For FL specimens (Fig. 2i , Fig. 3c ), total RNA and genomic DNA were prepared and stored using TRIzol and RNeasy Midi Kits (Qiagen). Sufficient nucleic acid was confirmed for 80% of archival FL specimens after quality control assessment of a subset of these patients. Total RNA from FL samples was linearly amplified (3′ IVT Express, Affymetrix) prior to microarray hybridization. For all above samples, total cellular RNA (at least 300 ng) was assessed for yield (NanoDrop 2000, Thermo Scientific), and quality (2100 Bioanalyzer, Agilent), and cRNA was hybridized to HGU133 Plus 2.0 microarrays (Affymetrix) according to the manufacturer’s protocol.
Two additional cohorts of PBMCs were analyzed in this study (Fig. 3a,b ). For the first cohort (n = 20 subjects; Fig. 3a ), PBMCs (~1×106 viable cells per mL) were collected in 1 mL TRIzol (Invitrogen) and stored at −80 °C until use. Total RNA was isolated according to the TRIzol protocol (Invitrogen). Total RNA yield was assessed using the Thermo Scientific NanoDrop 1000 micro-volume spectrophotometer (absorbance at 260 nm and the ratio of 260/280 and 260/230). RNA integrity was assessed using a Bioanalyzer NANO Lab-on-a-Chip instrument (Agilent). Biotinylated, amplified antisense complementary RNA (cRNA) targets were prepared from 200 to 250 ng of total RNA using the Illumina RNA amplification kit (Life Technologies), and 750 ng of labeled cRNA was hybridized overnight to Human HT-12 V4 BeadChip arrays (Illumina). The arrays were then washed, blocked, stained and scanned on an Illumina BeadStation 500 following the manufacturer’s protocols. GenomeStudio software version 1.9.0 (Illumina) was used to generate signal intensity values from the scans. For the second cohort (Fig. 3b ), PBMCs (1.4×106 to 4.0×106 cells per mL) from six healthy male adults were isolated and prepared as described in Supplementary Note and then frozen at −80 °C until use. Total cellular RNA ≥300 ng) was isolated from these six subjects along with viably preserved PBMCs from patient 4 (Supplementary Fig. 4c ) using RNeasy Mini Kit (Qiagen) and assessed for yield (NanoDrop 2000, Thermo Scientific), and quality (2100 Bioanalyzer, Agilent). Total RNA was linearly amplified (3′ IVT Express, Affymetrix), and cRNA was hybridized to HGU133A microarrays (Affymetrix) according to the manufacturer’s protocol.
Two additional cohorts of PBMCs were analyzed in this study (
