Computational protocol: Two amino acid mutation disrupts RORγt function in Th17 differentiation but not thymocyte development

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Protocol publication

[…] To measure gene expression in the thymus of C57BL/6, RORγt−/− or RORγtM/M mice, two separate samples were collected on different days, and thymocytes from four (two male and two female) were pooled each day. For demonstrating the gene expression profile regulated by wild-type RORγt or RORγtM/M in in vitro-differentiated Th17 cells, CD4+ T cells were enriched from RORγt−/− mice, transduced with retrovirus containing empty vector, wild-type RORγt or RORγtM/M, then polarized under Th17 conditions for three days. Cells were processed for RNA isolation (Qiagen). Quality verification, library preparation, and sequencing were performed at City of Hope Integrative Genomics Core facility. Samples were sequenced on an IlluminaHiSeq 2000 using 75-bp paired end reads. Reads were aligned to the mm9 mouse genome using TopHat version 2.1.0 and Bowtie version 1.1.1. Differential expression was computed with Cufflinks version 2.2.1. Differentially expressed genes were filtered by significance at FDR-adjusted P < 0.05, and the ratio of fragments per kilobase of transcript per million mapped reads (FPKM) were at least two-fold changes between two of the populations. Clustering and heatmapping were performed using hierarchical clustering in R with pheatmap package version 1.0.8. For heat maps, expression values were scaled per gene. [...] 2×107 cells were incubated with 1% formaldehyde, to cross-link proteins with chromatin, for 5 min at room temperature. 125 mM glycine was added to stop the cross-linking reaction. To shear genomic DNA into 200–500 bp fragments, cell lysates were sonicated using a water-bath sonicator (Covaris S200). Cell lysates were centrifuged (12000 g, 10 min) and incubated with FLAG antibody (M2, Sigma-aldrich, listed in Life Sciences Reporting System item 9) or IgG controls, and magnetic protein A/G beads (Millipore). After extensive washing, DNA was eluted followed by reversion of the protein–DNA cross-linking. DNA was recovered for DNA-seq or qRT-PCR to quantify specific DNA fragments that were precipitated. Primers used in qRT-PCR were listed in . Samples were sequenced on an IlluminaHiSeq 2000 using 75-bp paired end reads. Reads were aligned to the mm9 mouse genome using Bowtie version 1.1.1. Differential peaks were computed with MACS version 1.4.2 and bedtools version 2.17.0. Neighboring genes in the mm9 mouse genome were searched using webtools Cistrome Analysis Pipeline (at website: Cistrome.org/ap). […]

Pipeline specifications

Software tools Bowtie, BEDTools, Cistrome
Application ChIP-seq analysis
Organisms Mus musculus