Illumina HiSeq 4000 sequencing; RNA-seq of bone marrow neutrophils from wild-type mice (C57BL/6) and CD40LG knockout mice.
RNA-seq of bone marrow neutrophils from wild-type mice (C57BL/6) and CD40LG knockout mice.
Bone marrow cells were harvested aseptically from both femur and tibia bones and processed immediately for analysis. Mouse bone marrow neutrophils were purified by negative selection on an autoMACS cell separator using a neutrophil isolation kit (Miltenyi Biotec,  Cologne, BG) and following the manufacturer's instructions. The cell purity obtained using this procedure was verified by FACS staining of purified cells using antibodies specific to CD11b and Ly6G and was routinely found to be 90-95%.  After purification, RNA from bone marrow neutrophils was isolated by TRIZOL reagent (Invitrogen,  Carlsbad, CA) according to the manufacturer's instructions. When necessary, cells obtained from two different mice of the same group and processed simultaneously were pooled to obtain the number of cells required to perform the sequencing. RNA integrity and concentration were assessed using the Agilent 2100 Bioanalyser RNA Nanochip. Samples with RNA integrity number (RIN) ≥ 8 were used for thr transcriptome analysis.  cDNA libraries were obtained using the Illumina CBot station, and HiScanSQ was performed using the NEBNext Ultra Sample Preparation Kit (Illumina Inc, San Diego, CA) according to each manufacturer's instructions.