GSM9314922: Neutrophils, Donor Siglec14-NULL, unstimulated; Homo sapiens; RNA-Seq
Peripheral blood neutrophils (PBNs) from the healthy donor and Siglec14-NULL donor were isolated by negative sorting with magnetic beads. Freshly isolated PBNs were resuspended into phenol red- and serum-free RPMI and plated at a 0.5x106/ml concentration. They were then cocultured alone or in the presence of live bacteria at a 50:1 bacteria: neutrophil ratio. Reverse transcription was performed on mRNA captured by BD Rhapsody™ Cell Capture Beads usingthe BD Rhapsody™ cDNA Kit, following the manufacturer's instructions. This step generatedbead-bound cDNA via oligo(dT)-primed reverse transcription. Whole transcriptomeamplification (WTA) was then carried out using the BD Rhapsody™ WTA Amplification Kit,which amplifies 3ʹ end transcript fragments through targeted PCR to produce whole-transcriptome libraries. Purification steps were conducted using SPRIselect magnetic beadsaccording to the recommended ratios for each step of the protocol. Final libraries werequantified using the Qubit™ dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific), and39size distribution was assessed on an Agilent 2100 Bioanalyzer using the High Sensitivity DNAkit (Agilent Technologies).