Transcriptome Sequencing of mice eosinophils upon Listeria monocytogenes (L.m.) infection

Mature eosinophils were differentiated from mouse bone marrow progenitors We performed transcriptome sequencing on Listeria monocytogenes infected eosinophils and resting eosinophils to shed light on the transcriptional changes of cytokines and chemokines. 3×10^6 Eosinophils were infected with Listeria monocytogenes for 8 hours. Resting and infected eosinophils were used for RNA extraction, and sequencing. Total RNA was isolated using RNeasy mini kit (Qiagen, Germany). Strand-specific libraries were prepared using the TruSeq® Stranded Total RNA Sample Preparation kit (Illumina, USA) following the manufacturer’s instructions.Ribosomal RNA was removed from total RNA using Ribo-Zero rRNA removal beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under 94℃ for 8 min. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then go through an end repair process, the addition of a single ‘A’ base, and then ligation of the adapters. The products are then purified and enriched with PCR to create the final cDNA library. Purified libraries were quantified by Qubit® 2.0 Fluorometer (Life Technologies, USA) and validated by Agilent 2100 bioanalyzer (Agilent Technologies, USA) to confirm the insert size and calculate the mole concentration. Cluster was generated by cBot with the library diluted to 10 pM and then were sequenced on the Illumina NovaSeq 6000 (Illumina, USA).