NextSeq 550 paired end sequencing; RNA sequencing of neutrophils residing in bone marrow, peripheral blood and lung
RNA sequencing of neutrophils residing in bone marrow, peripheral blood and lung
4T1 cells (5×105 cells) were re-suspended in 50 µl growth factor-reduced Matrigel (Corning) and injected into the fourth mammary fat pads of female syngeneic BALB/cJ mice (8-week-old, n = 3). At day 10 (pre-metastatic stage), the mice were euthanized and then CD45+CD11b+Ly6GhighLy6Cmed neutrophils from bone marrow (BM), peripheral blood (PB) and lung were isolated by fluorescence-activated cell sorting. 4T1 cells were injected into the fourth mammary fat pads of female syngeneic BALB/cJ mice (8-week-old, n = 3). At day 10 (pre-metastatic stage), the mice were euthanized and then CD45+CD11b+Ly6GhighLy6Cmed neutrophils from bone marrow (BM), peripheral blood (PB) and lung were isolated by fluorescence-activated cell sorting. Total RNA was isolated from neutrophils using the miRNeasy Mini kit (Qiagen). Stranded libraries were prepared using the KAPA mRNA HyperPrep Kit (KAPA Biosystems): 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. Libraries were checked for quality and concentration using the DNA 1000 assay (Agilent Technologies) and quantitative PCR (KAPA Biosystems), according to the manufacturers' instructions.