Computational protocol: Blimp1/Prdm1 Functions in Opposition to Irf1 to Maintain Neonatal Tolerance during Postnatal Intestinal Maturation

Similar protocols

Protocol publication

[…] Small intestines were dissected, flushed with PBS, cut into small pieces and cross-linked with 1% formaldehyde in PBS for 15 min at 4°C followed by 35 min at 25°C [] and subsequently processed for ChIP using either 10 μg of mouse anti-GFP IgG2a (clone 3E6, A11120; Invitrogen), polyclonal rabbit anti-Irf1 antibody (M-20, Santa Cruz, SC-640) or as a control, normal rabbit IgG (Santa Cruz, SC-2027) as described previously []. The DNA samples were multiplexed and sequenced using two lanes on an Illumina HiSeq 2000 sequencer. Duplicate test (GFP ChIP of Prdm1 BEG/BEG, or Irf1 ChIP of WT and Villin-cre conditional Blimp1 mutants, []) samples or individual negative control (GFP ChIP of wild type or, normal rabbit IgG ChIP) and input samples were analyzed.Sequence reads were mapped to the mm9 mouse genome release with Stampy using default parameters []. Peak calling was performed with MACS1.4.2 [,], using default parameters to call areas of enrichment in ChIP samples over input. Regions of enrichment detected in negative controls samples were removed from subsequent analysis. GFP ChIP peaks called in wild type samples were subtracted from GFP peaks called in BEG/BEG samples. Similarly, normal rabbit IgG ChIP peaks were subtracted from Irf1 peaks. The overlapping peaks in duplicate ChIP samples were then identified and the core region of overlap was further analyzed. The genomic distribution of ChIP-seq peaks compared to gene annotations was determined using CEAS []. Genes of Ensembl release 67 with proximal Blimp1 or Irf1 binding were identified using custom Perl scripts. De novo identification of motifs within ChIP-seq peaks was performed using MEME suite tools []. Functional annotation of ChIP-seq peaks was performed with GREAT version 2.0.2 using the basal plus extension rule, annotating genes within 5 kb of transcription start sites initially or within 25 kb when no proximal gene in known to exist []. Regions of overlap between the ChIP-seq peaks identified in present study with other published datasets were compared using custom Perl scripts. Peak regions in human datasets were first converted to mm9 using the UCSC liftOver function. The association between Blimp1 binding (± 5kb of TSS) and genes differentially expressed in embryonic Prdm1 -/- small intestine [] (Gene Expression Omnibus database, www.ncbi.nlm.nih.gov/geo, accession no. GSE29658) was calculated by chi-square test.For comparative Irf1 ChIP-seq analysis reads mapping within identified peaks were counted using HTSeq-count []. TMM normalization factors and tagwise dispersions were then computed, and differential occupancy between sites in mutant and wild type determined by exact test using edgeR [,]. False discovery rates were calculated using the Benjamini and Hochberg method. Sites differentially bound with an FDR ≤ 0.05 were considered significant. […]

Pipeline specifications

Software tools Stampy, CEAS, MEME Suite, UCSC LiftOver, HTSeq, edgeR
Application ChIP-seq analysis
Organisms Mus musculus, Homo sapiens