RNA-Seq of medullary neutrophils
Synthesis of cDNA was accomplished by SMART-Seq v4 Ultra Low Input RNA kit for sequencing (Takara Bio, Gothenburg, Sweden). One nanogram of total RNA was used for first-strand synthesis. Full-length cDNA was generated via template switching (SMART) and PCR was performed according to the manufacturers protocol. Quantification of cDNA was done using the Qubit dsDNA HS Assay kit (Thermo Fisher Scientific, Langenselbold, Germany). Libraries for NGS were prepared from one nanogram cDNA using the Nextera XT Library Preparation kit (Illumina, San Diego, USA). After tagmentation, barcodes were ligated and PCR was performed. With the exception that final elution after bead purification was carried out with 34 L of resuspension buffer, sample preparation was performed according to the manufacturers protocol (Illumina, San Diego, USA). Libraries were quantified by Qubit dsDNA HS Assay kit (Thermo Fisher Scientific, Langenselbold, Germany) and quality was assessed using the High Sensitivity NGS kit on a Fragment Analyzer (Agilent, Waldbronn, Germany). Sequencing of libraries was performed on NextSeq2000 (Illumina, San Diego, USA), resulting in 40 million 100-bp single-end reads per sample.