Similar protocols

Protocol publication

[…] ed out by the Australian Genome Research Facility., ChIP was carried out using extracts of TWIST1-expressing MDCK cells . Cross-linking in 1% formaldehyde, lysis and sonication were carried out as described . Extracts were pre-cleared by incubation with A/G magnetic beads (Dynal) for 3 hrs and incubated with an anti-TWIST1 monoclonal antibody (Abcam ab50887) overnight at 4 °C, before adding blocked beads and subsequent washing steps in RIPA buffer, RIPA/NaCl buffer and LiCL buffer . Sequencing was carried out by the Australian Genome Research Facility., Raw microarray data were log2 transformed, quantile normalized and differential expression analyzed using the Linear Models for Microarray (LIMMA, implementation within Gene Pattern. Differentially expressed genes were filtered on a false discovery rate (FDR) of 0.05., For ChIP-Seq data, 50 bp reads were trimmed using Cutadapt , filtered by quality score and aligned to the CanFam3 dog genome using bowtie2 as described . Peaks were called using MACS2 and IDR analysis performed using an IDR cut-off of 0.05. Peak coordinates from two replicates were merged, using the most extreme start and end positions of the two replicates. The equivalent mouse genome (mm10) peak genomic locations were determined using Liftover (NCBI) annotated using the R library ChipSeeker., ., ., We thank the staff of the CMRI BioResources Unit for animal husbandry., Our work was supported by the National Health and Medical Research Council (NHMRC), Australia (Grant ID 1066832), the Australian Research Council, Australia (Grant DP 1094008) and Mr James Fairfax. HB was supported by an NHMRC Biomedical Postgraduate […]

Pipeline specifications

Software tools limma, cutadapt, Bowtie2