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Genome-wide sequential ChIP (ChIP-reChIP) will also identify the genomic sites at which different transcription factor complexes are positioned.
ChIP-qPCR and sequential ChIP experiments validate the ChIP-chip data and support the co-enrichment of trimethylated H3K4 and H3K27 on a subset of genes.
Sequential ChIP (Seq-ChIP) was performed according to the protocol found in Supplementary Protocol S.1.
To directly test whether RNAPII and PRCs simultaneously coassociate at PRC-repressed chromatin, we used sequential ChIP (re-ChIP).
Results from sequential ChIP (re-ChIP) show that the two complexes have similar accessory-factor compositions but differ in their preference for the recruitment of coactivators and corepressors.
The signals from each array element attributed to the sequential-ChIP experimental datasets were obtained by subtracting signal for each sequential ChIP control from the sequential-ChIP values in the ChIP channel.
Sequential ChIP assays were conducted to distinguish between these possibilities.
The sequential ChIP experiments were performed as previously described [73] with modifications.
The sequential ChIP assay, therefore, suggests H3K4me3/K27me3 bivalency on the sox2 and sox3 promoters.
The sequential ChIP assays were conducted as described in Shang et. al. with some modifications [28].
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For sequential-ChIP (ChIP for histone modifications in both rounds) and sequential-ChIP controls (ChIP for histone modification in first round followed by ChIP for rabbit IgG in the second round), signal intensities in both channels were normalized independently and averaged for bioreplicates (based the median input signals), and then normalized between experimental and control datasets.
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