2.8. RNA extraction, cDNA library preparation, and sequencing

Mice immunized with yPvCSP-All_CT epitopes or yNLP-PvCSP_CT adjuvanted with Poly I:C, or Poly I:C alone had their spleens excised two weeks after the immunization regimen as well as naive mice. Splenic B-cells were purified using MagniSort ™Mouse B cell Enrichment (ThermoFisher Scientific), resuspended in RNAlater solution (ThermoFisher Scientific), and stored at -80°C until use. Total RNA was extracted using the Quick - RNA Miniprep kit (Zymo Research, USA) following the manufacturer’s instructions. RNA integrity was verified for each sample using the Agilent 2100 BioAnalyzer and Agilent RNA 6000 Nano Chips (Agilent). mRNA preparation was performed using the rRNA depletion technique with the Agilent DNA 1000 kit and Agilent 2100 BioAnalyzer equipment. cDNA library preparation and sequencing were conducted by Quick Biology Inc (Pasadena, CA, USA) using the HiSeq 4000 equipment, generating approximately 24 million reads.