Illumina NovaSeq 6000 paired end sequencing; Comparison of coding RNA expression in steady-state and in response to IL33 and IL5 stimulation in bone marrow eosinophils from IL5-deficient and wild-type littermates
Comparison of coding RNA expression in steady-state and in response to IL33 and IL5 stimulation in bone marrow eosinophils from IL5-deficient and wild-type littermates
Single-cell mouse bone marrow suspensions were recovered from hind leg bones (pelvis, tibia, femur). Bones were flushed with 10mL of ice-cold PBS containing 10mM EDTA and passed through a cell strainer (70µM). The recovered single-cell suspension was lysed in lysis buffer at room temperature for 5 minutes and washed with ice-cold PBS twice. Mouse bone marrow cells were stained with anti-CCR3-APC (BioLegend, J073E5), anti-Siglec-F-PE (BD, E50-2440), and green nucleic acid stain (BD Biosciences, 565799). Absolute eosinophil counts were quantitated using CountBright absolute counting beads for flow cytometry (ThermoFisher, C36950). Bone marrow eosinophils (CCR3+ Siglec-F+ cells) were sorted into TRIzol (ThermoFisher, 15596026) using a BD FACSAria III (BD Biosciences) at a purity of 90 per cent or higher and stored at -80°C for downstream RNA applications. Mouse bone marrow eosinophils (purity≥95%) were cultured for 4 hours at 37°C and 5% CO2 in culture medium with additional 100ng/mL purified IL33 (BioLegend, 580504), and 10ng/mL purified IL5 (Peprotech, 215-15). Stimulated mouse bone marrow 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 10,000xg 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