Similar protocols

Protocol publication

[…] Library preparation and mRNA sequencing were performed as previously described (). The results were confirmed by qRT-PCR for at least 30 independent genes. Gene functional annotation was performed using DAVID (http://david.abcc.ncifcrf.gov/).For ChIP-seq, mouse livers, freshly isolated or snap frozen in liquid nitrogen, were homogenized by douncing and fixed with 1% PFA for 10 min and fixing was stopped by adding glycine at a final concentration of 0.125 M. Alternatively, for TAF3 and for TBP ChIP-seq, chromatin was fragmented by MNase I digestion as follows. Fixed nuclei were resuspended in equal volume of MNase buffer (50 mM Tris–HCl, pH 8.0, 15 mM NaCl, 5 mM CaCl2, 60 mM KCl) and treated with MNase (#MO247S; NEB Ipswich, MA) at 37°C for 10 min. The reaction was stopped by addition of EDTA to a final concentration of 20 mM. Chromatin was incubated with 0.3% SDS (5 min on ice) and with 1% Triton X-100 (5 min on ice) and centrifuged at 14,000 rpm. ChIP was performed overnight with 50 μg of chromatin and 5 μg of the following antibodies: anti-Pol II (sc-9001 X), anti-TFIIB (sc-225 X), anti-TFIIE (sc-6935 X), anti-HNF4A (sc-8987 X), anti-CDK9 (sc-8338 X), anti-HNF6 (sc-13050 X), anti-CEBPA (sc-9314 X), anti-FOXA2 (sc-6554 X) from Santa Cruz (Santa Cruz, CA), anti-TBP (ab28175) from Abcam (Cambridge UK), anti-CTCF (07–729) and anti-H3K4me3 (04–745) from Millipore (Billerica, MA), anti-TAF3 (IGBMC, in house). ChIP-seq libraries were prepared as previously described and sequenced on the Illumina Hi-seq2500 as single-end 36-base reads (). Peak detection was performed using the MACS software () using the no antibody ChIP as negative control. Data sets were normalised for the number of unique mapped reads for subsequent comparisons. Global clustering analysis and quantitative comparisons were performed using seqMINER (http://bips.u-strasbg.fr/seqminer/) () and R (http://www.r-project.org/) and visualized using ggplot2 package. Genomic region annotation was performed with either seqMINER or GREAT (http://bejerano.stanford.edu/great/public/html/). Overlap between HNF4A-binding sites that are enhanced or depleted in TAF4-mutant liver and CRMs was performed by intersecting the corresponding genomic coordinates. […]

Pipeline specifications

Software tools DAVID, seqMINER, Ggplot2
Applications Miscellaneous, ChIP-seq analysis
Organisms Mus musculus, Homo sapiens
Diseases Precancerous Conditions