Computational protocol: Canonical Wnt signalling regulates nuclear export of Setdb1 during skeletal muscle terminal differentiation

Similar protocols

Protocol publication

[…] Cells were grown on glass coverslips, which had been coated with collagen type I (Sigma) for 1 h at 37 °C and rinsed with PBS. C2C12 cells were fixed with 4% formaldehyde (FA; Sigma), incubated with 0.1 m glycine for 5 min to quench FA, permeabilised with 0.2% Triton-X-100 and blocked with PBS containing 2% donkey serum (Jackson Immunoresearch) for 1 h at RT. Primary and secondary Abs were diluted in PBS containing 2% donkey serum and 0.2% Tween and incubated overnight at 4C° or 1 h at RT, respectively. DNA was stained with 1 μg ml−1 DAPI (Sigma). Coverslips were mounted with Fluorescence Mounting Media (Dako). Primary myoblasts and fibres were processed in the same way; except blocking was done with 5% heat inactivated serum in PBS and DNA were counterstained with Hoechst (Life Technologies).Microscopy was performed using the Axioplan 2 (Zeiss). Images were taken with the Cool Snap fx camera (Photometrics) and analysed with Metamorph software. Confocal microscopy was performed using the LM710 microscope (Zeiss, Marly le Roi, France). Confocal images were analysed with ZEN 2011 software. Light microscopy was performed using the Leica DMIL LED microscope. All images were processed with ImageJ software (http://rsbweb.nih.gov/ij/) to identical contrast and brightness. [...] Sequenced reads were demultiplexed to attribute each read to a DNA sample and then aligned to reference mouse genome mm9 with bowtie (-n 2 -e 70 -l 50 --maxbts 125 -k 1 -m 1). After PCR duplicates were removed, Setdb1 and H3K9me3 enrichments were analysed over their respective control (Input DNA) by the software MACS 1.4 as previously described []. The P-value threshold was 10e-3, and the shift size 50 pb (-shiftsize 50 -no model). For H3K9me3 ChIP-seq an additional peak caller was used: SICER v 0.0.1 (e-value <0.01) with W200 and G1000 parameters. Setdb1 ChIP-seq peaks were determined on Setdb1 ChIP-seq and only binding sites with P-values <10e-5, FDR<1% and fourfold enrichment over the control were retained. For genome annotation we used Homer software v4.7 []. For bound genes, we used GREAT software (v3.0). Plots were generated with ngsplot package []. Genomatix software was used for Gene Ontology analyses. Hypergeometric test has been performed in R. [...] Sequencing was performed on a HiSeq 2500 (Illumina) in a 51 bases single read using a HiSeq SR Cluster kit v4 cBot HS (Illumina; # GD-401-4001) and a HiSeq SBS kit v4 HS 50 cycles (Illumina; # FC-401-4002). Sequences were demultiplexed using the Illumina pipeline (Gerald, included in CASAVA version 1.8) giving FASTQ formatted reads. Those reads were cleaned from adapter sequences and sequences of low quality using an in-house programme (https://github.com/baj12/clean_ngs). Sequences with a minimum length of 25 nucleotides were considered for further analysis. Tophat (version 1.4.1.1, default parameters) was used to align to the reference genome (mm9). HTseq-count (parameters: -m intersection-nonempty, -s yes, -t exon) was used for counting genes [, ]. Dseq2 package [] was used for statistical analysis. […]

Pipeline specifications

Software tools BaseSpace, TopHat, HTSeq
Application RNA-seq analysis
Organisms Mus musculus