S1 flow cell

After PicoGreen quantification and quality control by Agilent BioAnalyzer, 500 ng aliquots of genomic DNA were sheared using a LE220-plus Focused-ultrasonicator (Covaris catalog #500569). Sequencing libraries were prepared using the KAPA Hyper Prep Kit (Kapa Biosystems KK8504) with modifications. DNA libraries were subjected to size selection by mixture with 0.5 vol of aMPure XP beads (Beckman Coulter catalog # A63882) after post-ligation cleanup. Libraries were not amplified by PCR and were pooled equivolume for sequencing. Samples were run on a NovaSeq 6000 in a 150-bp/150-bp paired-end run using the NovaSeq 6000 SBS v1 Kit and an S1 flow cell (Illumina). The average number of read pairs per sample was 10 million.

Aliquots of total RNA (250 ng) from peripheral blood B cells were used to generate poly-adenylated RNA libraries with TruSeq Stranded Total RNA Ribo-Zero Human Gold kit (Illumina, San Diego, CA). Samples were indexed with NEXTfle-96 RNA-seq Barcodes (Bioo-scientific, Austin, TX) and 75 bp paired-end sequencing was performed on NovaSeq 6000 platform using S1 flow cell (Illumina) in the NIEHS Epigenomics and DNA Sequencing Core Laboratory. FASTQ files containing 26–119 million raw sequencing reads were aligned to hg38 using STAR and gene counts were generated with featureCounts using the GENCODE version 39 annotation. Count matrix data were then imported to Partek Flow (Partek Inc., St. Louis, MO) and quantification of transcript expression and differential expression analyses were performed using DESeq adjusting for covariates as done in methylation (age, sex, race, BMI, 5 cell-type proportions, with or without naïve B cell proportions). DEGs were determined between smokers and nonsmokers with a cutoff for significance at p < 0.05 and/or FDR-adjusted p < 0.01. Controlled hierarchical cluster analysis by smoking status generated heatmaps showing a structure of DEG expression trends and partition of DEGs into different clusters using Partek Flow. RNA-seq raw data are deposited in GEO (accession number: GSE220113).

For the 10X Genomics assembly, HMW genomic DNA was isolated from whole blood stored in the PAXgene proprietary media using the Nanobind CBB Big DNA kit (Circulomics, Inc.) and short fragments filtered out using the Circulomics Short Read Eliminator kit. Genomic DNA concentration and purity were assessed with a Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Capillary electrophoresis was carried out using a Fragment Analyzer (Agilent Technologies) to ensure that the isolated DNA had a minimum molecule length of 40 kb. Genomic DNA was diluted to ∼1.2 ng/μl and libraries were prepared using Chromium Genome Reagents Kits Version 2 and the 10X Genomics Chromium Controller instrument fitted with a micro-fluidic Genome Chip (10X Genomics). DNA molecules were captured in Gel Bead-In-Emulsions (GEMs) and nick-translated using bead-specific unique molecular identifiers (Chromium Genome Reagents Kit Version 2 User Guide) and size and concentration determined using an Agilent 2100 Bioanalyzer DNA 1000 chip (Agilent Technologies). Libraries were then sequenced on an Illumina NovaSeq 6000 System using an S1 flowcell, following the manufacturer’s protocols (Illumina) to produce >95× read depth using paired-end 150 bp reads. The reads were assembled into phased pseudo-haplotypes using Supernova Version 2.0 (10X Genomics).