Neutrophil RNA Extraction and Sequencing

Neutrophil RNA was extracted as described previously [22 (link)] using TRIzol, according to the manufacturer’s instructions. Trace DNA was removed by DNase I (Life Technologies, Carlsbad, CA) treatment. RNA was purified using the RNeasy MinElute Cleanup Kit (Qiagen, Venlo, Limburg, Netherlands) and suspended in RNase-free water. The purity and concentration of RNA in each sample were measured by absorbance at 260 nm and 280 nm on a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA), and 200 ng to 400 ng of RNA was reserved for sequencing. Precise RNA concentration was measured via the Quant-iT RiboGreen Assay (Invitrogen, Carlsbad, CA) with a TBS-380 Fluorometer (Promega, Madison, WI), and the quality of the RNA samples was measured with an Agilent 2100 BioAnalyzer RNA 6000 Pico Chip (Agilent, Las Vegas, NV). RNA samples to be sequenced had acceptable purity (260/280 ratio of ~ 1.8 or greater, range: 1.76–2.12) and integrity (RIN of ~ 5 or greater, range: 4.5–9.1) prior to RNA sequencing.