Tapestation 4200

Bulk RNA-seq was performed on purified CD14⁺ monocytes after sorting. In brief, after sorting, 5,500 cells were washed, resuspended in 350 μL chilled Buffer RLT (Qiagen, 79216) supplemented with 1% beta-Mercaptoethanol (Sigma, M3148–25ML), vortexed for 1 minute, and immediately frozen at −80C. RNA was isolated using the RNeasy Micro kit (Qiagen, 74004) with on-column DNase digestion. RNA quality was assessed using an Agilent Bioanalyzer and total RNA was used as input for cDNA synthesis using the Clontech SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio, 634894) according to the manufacturer’s instructions. Amplified cDNA was fragmented and appended with dual-indexed bar codes using the NexteraXT DNA Library Preparation kit (Illumina, FC-131–1096). Libraries were validated by capillary electrophoresis on an Agilent 4200 TapeStation, pooled at equimolar concentrations, and sequenced on an Illumina NovaSeq6000 at 100SR, yielding 20–25 million reads per sample.

Libraries from 5–10 ng ChIP DNA were prepared using KAPA Hyper Library Preparation Kit (KAPABIOSYSTEMS, #KK8504). Precipitated DNA was quantitated on the Qubit® 4 Fluorometer (Invitrogen). Samples were end-repaired, 3’ ends-adenylated and barcoded with multiplex adapters, followed by size selection with Ampure XP beads (BECKMAN COULTER, #A63881) and PCR amplification. Samples were validated on the Agilent Tapestation 4200, normalized, pooled, and run on the Illumina NextSeq 500 using 75 cycle SBS v2.5 reagents. Analysis for ChIP-sequencing was conducted using the UTSW BICF ChIP-seq Analysis workflow (31 (link)). Briefly, raw fastq files were trimmed by TrimGalore and then aligned to human reference genome (hg38) using BWA (32 (link)). Low-quality reads and duplicate reads were removed from aligned files using Sambamba (33 (link)) and Samtools (34 (link)). Model-based Analysis of ChIPSeq (MACS) (35 (link)) software tool (v.2.1.2) was used to call peaks from the ChIP-sequencing data. All peaks were annotated using ChipSeeker (36 (link)) and differential binding activity was calculated using DiffBind (37 (link)). ChIP peaks were visualized using IGV (https://igv.org/).

Mouse alveolar macrophages were isolated via FACSorting at indicated time points. Approximately 100,000 cells were sorted into MACS buffer, immediately pelleted and lysed in RLT Plus buffer supplemented with 2-mercaptoethanol (Qiagen). RNA was isolated using RNeasy Plus kit with genomic DNA removal step. RNA quality was assessed on TapeStation 4200 instrument (Agilent), all samples had RNA integrity number (RIN) over 7. RNA-seq libraries were prepared from 100 ng of total RNA, starting with poly(A) enrichment and followed by NEB Next RNA Ultra I chemistry. Libraries were quantified and assessed on Qubit fluorimeter (Invitrogen) and TapeStation 4200, correspondingly, multiplexed and sequenced on NextSeq 500 instrument (Illumina), 75 bp, single end reads, to the average sequencing depth of 6×10⁶ reads per sample. Over 94% of reads had Q score over 30. Reads were demultiplexed and mapped to mm10 version of the mouse genome using TopHat2 aligner and mapped to the genomic features using HTSeq and counts processed using edgeR package to estimate differentially expressed genes. FDR p value less than 0.05 was used to identify differentially expressed genes. K-means clustering was performed using GENE-E. Gene ontology analysis was performed using GOrilla on two unranked gene lists. The RNA-seq dataset is available at GEO: GSE98731.