Illumina NovaSeq 6000 paired end sequencing; RNA-seq of BM neutrophils after cultured with lung CD140a+ MCs
RNA-seq of BM neutrophils after cultured with lung CD140a+ MCs
BM neutrophils were isolated from six 10-week old female mice (C57BL/6J) and co-cultured with lung CD140a+ MCs overnight. Then neutrophils were harvested for RNA-seq. Cells were lysed and homogenized in TRIzol Reagent (ThermoFisher), then RNA was isolated using the miRNeasy Mini kit (Qiagen), according to manufacturers' protocols, including the optional DNase digest step.  Libraries were constructed using the KAPA mRNA HyperPrep Kit (Roche Sequencing and Life Science), according to the manufacturer's protocol.  Briefly, the protocol entails isolation of polyA containing mRNA using oligo-dT magnetic beads, RNA fragmentation, first and second strand cDNA synthesis, ligation of Illumina-specific adapters containing a unique barcode sequence for each library, and PCR amplification.  The quality and concentration of the libraries were assessed using the D5000 ScreenTape (Agilent Technologies) and Qubit dsDNA HS Assay, respectively, according to the manufacturers' instructions.