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Enriched regions of the genome were identified by comparing the ChIP sample with input using the MACS peak caller (Zhang et al., 2008) version 1.3.7.1.
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For Caco-2 CLIP samples with input RNA as control, we compared four different ranking and normalization strategies, including Raw, AVE-CLIP, AVE-INPUT, and INPUT (Materials and Methods).
However, the quality of sequence data dropped sharply for samples with input DNA <10 pg (Fig. 5).
AR-enriched genomic regions (binding sites) were identified by comparing the ChIPed samples with input sample using MACS algorithm [ 16] (1.4.0rc 2) and option of "-p 1e-10".
A similar trend was observed with the proportion of gap sizes (length of uncovered bases, Fig. 5 bottom panel) and chimeric reads (Fig. 4), where a sharp increase in these values was observed for samples with input DNA <10 pg.
Using these metrics, we show that the number of callable loci (high-quality bases) remained relatively high for samples with input DNA ranging from 2 ng to 10 pg.
The average computation for each sample time with input data points is (21).
8. Normalize this T molecule number in each RNA sample with the input of total RNA in the RT reaction.
For example, a sample with an input of 10 ng into the qPCR would have a QFI of 100% if all 3,030 haploid templates were available for amplification.
Most of the existing analysis tools are focused on the delineation of enriched sites from a single sample with optional "input control" [ 4].
Filtering out such pseudobinding sites is only possible by comparing the ChIP sample with an input sample, and this step is essential for the specificity of peak calling (Rozowsky et al. 2009).
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