Computational protocol: E2 multimeric scaffold for vaccine formulation: immune response by intranasal delivery and transcriptome profile of E2-pulsed dendritic cells

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Protocol publication

[…] Bone marrow-derived dendritic cells (BMDCs) were generated according to previously described protocols []. Briefly, bone marrow cells were collected by flushing tibias of C57BL/6 mice (Charles River Laboratory) with complete medium (RPMI 1640, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 % FCS). Cells were seeded in bacteriological Petri dishes in complete medium supplemented with 200 U/ml of recombinant murine granulocyte-macrophage colony stimulating factor (GM-CSF, Peprotech, NJ, USA) for 7 days. Immature cells were harvested and incubated or not for 1 more day with 50 μg/ml of LPS-free E2wt antigen at a final concentration of 2.5 × 106 cells/ml. Total RNA was extracted from untreated or E2-pulsed BMDCs using Tri Reagent (Sigma Aldrich) according to manufacturer's protocol. RNA integrity was assessed as described in Costa et al. []. Paired-end libraries (100 x 2 bp) prepared using TruSeq RNA Sample Preparation Kit (Illumina Inc., San Diego, CA) were sequenced on Illumina HiSeq2000 platform. About 200 million paired-end reads were sequenced. Quality was assessed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). TopHat version 2.0.10 [] was used to map reads on reference mouse genome (mm9) and RefSeq mouse transcripts annotation with default parameters. More than 95 % of sequenced reads uniquely mapped to mm9. Such reads were used for further analyses. Coverage files were produced using BEDTools and loaded on UCSC Genome Browsers to analyze gene-specific features. Gene expression was measured using Cufflinks 2 []. Cuffdiff and CummeRBund [] were used to identify differentially expressed genes using normalized expression values (FPKM, Fragments Per Kilobase of transcript and Million of mapped reads). An arbitrary threshold of 0.05 FDR (False Discovery Rate) and 1 FPKM in at least one condition was used to filter out differentially expressed genes. Gene ontology and pathway analysis were performed using DAVID (Database for Annotation, Visualization and Integrated Discovery) []. […]

Pipeline specifications

Software tools FastQC, TopHat, BEDTools, Cufflinks, CummeRbund, DAVID
Applications RNA-seq analysis, Transcriptome data visualization
Organisms Mus musculus, Human immunodeficiency virus 1