GSE172279
Bulk RNAseq of Sort-purified IV- Primary (1M) or Quaternary (4M) Tcirc (IV- CD69-/CD103-) or Trm (IV- CD69+/CD103+) Memory P14 cells isolated from Spleen or Mediastinal Lymph Nodes
Purpose: The goals of this study are to compare the phenotypic, transcriptomic, trafficking and functional atttibutes of Influenza-experienced circulating or resident memory phenotype cells from the spleen or lung-draining mediastinal lymph nodes. Results: Following read alignment, gene expression profiles were computed using featureCounts. Filtering and visualization of differentially expressed genes with a log2fold change >1.5<-1.5 and a p value of less than 0.05 were identified using Partek GS software and for some figures values from Partek were normalized and displayed using GraphPad Prism. Gene set enrichment and functional assignment were performed in DAVID bioinformatics resources and software from the Broad Institute. Overall design: B6 mice containing CD90.1 congenic Primary (1M) or Quarternary (4M) Memory P14 cells at 23-30 days post Intranasal Influenza (PR8-GP33) infection were IV labeled with CD45.2-BV450 3 minutes prior to sacrifice. Pooled splenocytes (6-10 samples/sort) or Mediastinal Lymph Nodes (15-25 samples/sort) were isolated, processed through a 70uM cell strainer, stained with APC-labeled -CD19, -NK1.1 and -CD4 and CD103-PE, CD69-PE-CY7, CD90.1-PerCP-eFluor710, Live/Dead-e780. Samples were negatively enriched with aAPC-beads and Macs columns, resuspended in MACS buffer with DNAse for subsequent sorting. Populations of IV- CD69-/CD103- (Tcirculating) and IV- CD69+/CD103+ (Tms) were sort purified and resuspended in TRIzol. Freshly sorted 1M or 4M P14s were (as indicated above) were re-suspended in TRIzol and RNA was purified using a RNAeasy kit (Qiagen) according to the manufacturer's protocol. RNA was assessed for purity and quality using an Agilent 2100 Bioanalyzer and RNA seq was performed using the single cell/low input library kit (NEB), full-length cDNAs were sequenced on the Illumina HiSeq 2500 High output platform using 2x150 Paired-end Libraries. The sequence reads quality was checked using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and were aligned to the mouse genome version mm10 using STAR aligner.