Illumina NovaSeq 6000 paired end sequencing; Comparison of coding RNA expression of blood eosinophils from mepolizumab- or omalizumab-treated severe asthmatic patients and from healthy controls
Comparison of coding RNA expression of blood eosinophils from mepolizumab- or omalizumab-treated severe asthmatic patients and from healthy controls
From every donor patient, we sampled 30mL of venous blood in BD Vacutainer EDTA tubes (BD Biosciences, 367525). Polymorphonuclear cells were isolated by double-layer density gradient centrifugation. Recovered cells were purified by negative selection immunomagnetic purification (Stemcell Technologies, 17956) following the manufacturer's instructions. Briefly, cells were washed twice with ice-cold PBS supplemented with 2% heat inactivated fetal bovine serum (FBS, Sigma-Aldrich) and 1mM EDTA. Cell suspension was sedimented at 300xg for 5 minutes and adjusted to 5x107 cells per mL. For every mL of sample, 50uL of antibody cocktail was added, and cells were incubated at room temperature for 5 minutes. An equal volume of RapidSpheres was added and samples were immediately incubated in an EasySep magnet at room temperature for 3 minutes. Pre-sort eosinophil viability was 95 per cent or superior as assessed by trypan blue exclusion. Human blood eosinophils were stained with anti-CCR3-FITC (REA574, Miltenyi Biotec), anti-CD3-PE (REA613, Miltenyi Biotec), anti-CD19-PE (REA675, Milenyi Biotec), anti-Siglec-8-APC (REA1045, Milentyi Biotec), and anti-CD16-APC-H7 (3G8, BD Biosciences). Staining reactions were performed at 4°C following a 5-minute incubation with Fc receptor antibodies to reduce non-specific binding (BD Biosciences, 564219). Eosinophils were sorted into TRIzol (ThermoFisher, 15596026) using a BD FACSAria III (BD Biosciences) at a purity of 95 per cent or higher and stored at -80°C for downstream RNA applications. For every mL of TRIzol, 200µL of chloroform was added, and the samples were vigorously mixed and incubated for 2 minutes at room temperature. Samples were centrifuged at 10,000xg for 15 minutes at 4°C to separate the phases. The RNA-containing upper aqueous phase was transferred to a new microcentrifuge tube containing 475µL of isopropanol and 2µL of glycoblue (ThermoFisher, AM9515). Samples were centrifuged at 10,000g for 15 minutes and supernatant was discarded. One volume of 75% ethanol was added and samples were centrifuged at 10,000g for 1 minute to precipitate the RNA and discard the supernatant. RNA pellet was resuspended in 40µL of DNase/RNase-free water for a 15-minute DNase treatment (Zymo Research, E1010). DNase treatment was followed by column-based RNA purification with the RNA Clean & concentrator-5 kit (Zymo Research, R1016). Briefly, 100µL of RNA binding buffer was added to every 50µL sample and mixed thoroughly. One volume of 100% ethanol was added and the sample was transferred into a Zymo-Spin IC column in a collection tube. Columns were centrifuged at 10,000 g for 30 seconds and flow-through was discarded. The column was washed once with RNA prep buffer and twice with RNA wash buffer, following the manufacturer's instructions. RNA was eluted in 10µL of DNase/RNase-free water and stored at -80°C. Purified RNA was quantitated and quality was assessed with Agilent RNA 6000 Pico (Agilent, 5067-1513). RNA integrity was assessed on a Agilent 2100 Bioanalyzer bioanalyzer chip (manufacturer) for the presence of 18s and 28s rRNA peaks. Full length cDNA was prepared from 500pg total RNA with SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio, 634889) following the manufacturer's instructions.Final cDNA quality was assessed using Agilent High Sensitivity DNA kit (Agilent, 5067-4626). cDNA libraries were prepared for sequencing using Nextera XT DNA library preparation kit (Illumina, FC-131-1024) using the manufacturer's instructions