Hiseq 2500 system rapid mode

After removing medium, we washed macrophages twice with cold PBS to get rid of floating cells. We washed THP-1 cells twice in PBS by centrifugation. We isolated total RNA using RNeasy Mini Kit together with QIAshredder (Qiagen). We removed the residual DNA by 30-min DNase treatment (TURBO DNA-free Kit, Ambion) at 37°C. Subsequently, we purified RNA with RNA Clean and Concentrator (Zymo Research), and checked its concentration and integrity using ribogreen assay and bioanalyzer (Agilent Technologies 2100 Bioanalyzer). We only processed RNA samples with RIN above 8. We prepared RNA libraries using the TruSeq Stranded mRNA Library Prep Kit (Illumina). In brief, we purified poly-A containing mRNA molecules by poly-T oligos attached to magnetic beads. We cleaved and fragmented the mRNA into small pieces, followed by reverse transcription of the first cDNA strand using random primers. We replaced dTTP with dUTP in the second strand synthesis by using DNA Polymerase I and RNase H, so as to track the strand information in the following steps. We ligated the cDNA fragments with the adapter and purified them. We enriched the molecules by PCR to generate the final cDNA library. We used HiSeq 2500 System Rapid mode (Illumina) for RNA-sequencing, giving rise to paired-end reads of 101 bp. We show total read numbers of each sample in Supplemental Table S4.