GSM8386352: Ctrl_EOS_2; Mus musculus; RNA-Seq
To isolate asthmatic lung cells, lungs were chopped into small fragments and enzymatically digested with Collagenase D and DNase I at 37 °C for 30 minutes. The enzyme reaction was stopped by adding EDTA. After vigorous resuspension, cells were filtered through 100 µm strainers, and red blood cells were lysed with ACK lysis buffer. Eosinophils were purified using fluorescence-activated cell sorting (FACS). Purified eosinophils were lysed with Qiazol and chloroform was added in a 5:1 ratio. Following centrifugation, the clear upper phase was collected, and RNA was precipitated by adding an equal volume of 100% isopropanol along with 20 µg of glycogen. After another round of centrifugation, the resulting pellet was washed with 75% ethanol and allowed to air-dry. Lastly, RNA was dissolved in nuclease-free water. Ribosomal RNAs were removed using NEBNext rRNA Depletion Kit v2, and the cDNA library was synthesized using NEBNext Ultra II Directional RNA Library Prep kit according to the manufacturer's protocols.