Illumina NovaSeq 6000 paired end sequencing; Transcriptomic response to ex vivo IL33 stimulation of blood eosinophils from mepolizumab- or omalizumab-treated patients
Transcriptomic response to ex vivo IL33 stimulation of blood eosinophils from mepolizumab- or omalizumab-treated patients
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 using a BD FACSAria III (BD Biosciences) at a purity of 95 per cent or higher. Culture medium consisted of sterile filtered IMDM supplemented with 20% heat inactivated FCS (Sigma-Aldrich), 1% non-essential amino acids (Sigma-Aldrich), 1mM sodium pyruvate (Sigma-Aldrich), 2mM l-glutamine (Sigma-Aldrich), 25mM HEPES (Sigma-Aldrich), 50µM -mercaptoethanol (Sigma-Aldrich), 100U/mL penicillin (Millipore). Human blood eosinophils (purity≥95%) were cultured for 6 hours at 37°C and 5% CO2 in culture medium with additional 100ng/mL purified IL33 (PeproTech, 200-33). Stimulated human blood eosinophils were sedimented at 300xg for 5 minutes, resuspended in TRIzol(ThermoFisher) and stored at -80°C for downstream RNA extraction.  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 10,000g 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