Computational protocol: Reciprocal regulation of the Il9 locus by counteracting activities of transcription factors IRF1 and IRF4

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Protocol publication

[…] Naive CD4+ T cells isolated from WT and Irf1−/− mice were cultured for 2 days under Th9 conditions in the presence of IFN-γ (5 ng ml−1). RNA was purified with the RNeasy Plus Mini Kit according to the manufacturer's protocol (Qiagen). RNA was quantified with a Qubit 2.0 fluorometer (Invitrogen) and the quality was assessed on a Bioanalyzer 2100 (Agilent) using a RNA 6000 Nano chip (Agilent). Samples with an RNA integrity number of >8 were used for library preparation. Barcoded mRNA-seq cDNA libraries were prepared from 400 ng of total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext Ultra RNA Library Prep Kit for Illumina according to the manual. Quantity was assessed using Invitrogen's Qubit HS Assay Kit and library size was determined using Agilent's 2100 Bioanalyzer HS DNA assay. Barcoded RNA-Seq libraries were onboard clustered using HiSeq Rapid SR Cluster Kit v2 using 8 pM and 50 bps were sequenced on the Illumina HiSeq2500 using the HiSeq Rapid SBS Kit v2 (50 Cycle). The raw output data of the HiSeq was preprocessed according to the Illumina standard protocol. Quality control on the sequencing data was performed with the FastQC tool (available at http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), as well as the comprehensive Qorts suite. Inspecting the produced reports, all samples were deemed of good quality for further processing. Short reads alignment was performed with the ENSEMBL Mus_musculus.GRCm38 chosen as the reference genome. The corresponding annotation (ENSEMBL v76) was also retrieved from the ENSEMBL FTP website (http://www.ensembl.org/info/data/ftp/index.html). The STAR aligner (version 2.4.0b) was used to perform mapping to the reference genome. Alignments were processed with the featureCounts function of the Rsubread package, using the annotation file, also used for supporting the alignment. Exploratory data analysis was performed with the pcaExplorer package. Differential expression analysis was performed with DESeq2 (version 1.12.3, https://www.ncbi.nlm.nih.gov/pubmed/25516281), setting the False Discovery Rate to 0.05 (ref. ). Gene expression profiles were plotted as heatmaps (colour-coded z-scores for each fragments per kilobase of exon per million fragments mapped value) with the R statistical software. GSEA was performed as previously described. […]

Pipeline specifications

Software tools FastQC, QoRTs, Subread, PcaExplorer, DESeq2, GSEA
Application RNA-seq analysis
Organisms Homo sapiens, Mus musculus