Computational protocol: RNA Seq and ChIP Seq reveals SQSTM1/p62 as a key mediator of JunB suppression of NF κB dependent inflammation

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Protocol publication

[…] For RNA-seq, total RNA was isolated from triplicate sets of human keratinocytes at 48h after transfection using RNAeasy column purification (Qiagen, Germantown, MD). cDNA library was prepared via oligo-dT-directed reverse transcription (Ambion, Grand Island, NY), and subject to deep sequencing on Illumina HiSeq2000 (50bp single-read sequencing) and analyzed at Duke Genome Sequencing & Analysis Resource. The analysis pipeline includes the initial QC to remove sequencing adaptors and low quality bases to facilitate mapping using fastx toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html). High quality reads were mapped to the human reference genome (hg19) using tophat (). The gene differential expression analysis was performed between control and treatment using cuffdiff with upper quartile normalization, multi-hit correction and 0.5% FDR(). Three biological replicates were used for statistical analysis of differential expression for each comparison. Pathway analyses were performed using WEB-based GEne SeT AnaLysis Toolkit (; ). SYBR green-based real-time RT-PCR was performed in Bio-Rad iCycler with primers listed in (). Ribosomal 18S RNA or GAPDH was used as an internal control. [...] ChIP was performed as described (). Briefly, primary keratinocytes cultured in eight 30-cm plates were scraped off the dish with PBS at about 60-80% confluence, and cross-linked with 1% formaldehyde for 8 minutes, and then treated with 1.25 M glycine for 5 minutes to stop crosslink. Cells were pelleted by centrifuging 5 minutes at 1500 rpm, and lysed with ChIP lysis buffer (Santa Cruz Biotechnology, Santa CruZ, CA) for cell nuclei isolation. DNA fragmentation was performed by sonication with Branson Sonifier. The fragmented DNA underwent IP with the antibody against JunB (Cell Signaling Technology, Beverly, MA) followed by purification with MinElute DNA kit (Qiagen) and library construction with the NEBNext® DNA library preparation kit (New England BioLabs). The library was sequenced using Illumina Hiseq 2000 platform and analyzed at Duke Genome Sequencing & Analysis Resource. The analysis pipeline includes the initial QC to remove sequencing adaptors and low quality bases to facilitate mapping using fastx toolkit (). High quality reads were mapped to the human reference genome (hg19) using bwa (), and genes lists were from the Ensemble Gene database (http://www.ensemble.org) and the RefSeq (www.ncbi.nlm.nih.gov/RefSeq). Distal promoter and terminator regions of the genes were defined as those sequences that mapped +/−2 kb upstream or +/− 50 kb from transcription “start” and “end” sites, respectively. Peak detection, annotation, de novo motif discovery and pathway analysis were performed using ChIPseeqer tool (), and visualized by UCSC genome browser (). Motif analysis was performed with FIRESeq as described(). ChIP-PCR was performed with primers listed in (). The raw data and additional information of ChIP-seq and RNA-seq are available from NCBI Gene Expression Omnibus (Accession number GSE63081). […]

Pipeline specifications

Software tools FASTX-Toolkit, BWA, ChIPseeqer
Databases GEO UCSC Genome Browser
Application ChIP-seq analysis
Organisms Mus musculus, Homo sapiens
Diseases Deficiency Diseases, Psoriasis