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yezhengSTAT authored and yezhengSTAT committed Dec 14, 2023
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E. coli DNA is carried along with bacterially-produced pA-Tn5 protein and gets tagmented non-specifically during the reaction. The fraction of total reads that map to the E.coli genome depends on the yield of epitope-targeted CUT&Tag and roso depends on the number of cells used and the abundance of that epitope in chromatin. Since a constant amount of pATn5 is added to CUT&Tag reactions and brings along a fixed amount of E. coli DNA, E. coli reads can be used to normalize epitope abundance across experiments.

The underlying assumption is that the ratio of fragments mapped to the primary genome to the E. coli genome (or other added DNA sequences if pA-Tn5 is purified and E.coli is not available anymore) is the same for a series of samples, each using the same number of cells. Because of this assumption, we do not normalize between experiments or cbetween batches of pATn5, which can have very different amounts of carry-over E. coli DNA. Using a constant C to avoid small fractions in normalized data, we define a scaling factor S as
The underlying assumption is that the ratio of fragments mapped to the primary genome to the E. coli genome (or other added DNA sequences if pA-Tn5 is purified and E.coli is not available anymore) is the same for a series of samples, each using the same number of cells. Because of this assumption, we do not normalize between experiments or batches of pATn5, which can have very different amounts of carry-over E. coli DNA. Using a constant C to avoid small fractions in normalized data, we define a scaling factor S as

$S = \frac{C}{(Fragments Mapped To E.coli Genome)}$

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## More resources about CUT&RUN and CUT&Tag data analysis

- [CUTRUNTools](https://bitbucket.org/qzhudfci/cutruntools/src/master/): [a flexible pipeline for CUT&RUN processing and footprint analysis](https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1802-4). CUT&RUNTools is a flexible and general pipeline for facilitating the identification of chromatin-associated protein binding and genomic footprinting analysis from antibody-targeted CUT&RUN primary cleavage data. CUT&RUNTools extracts endonuclease cut site information from sequences of short-read fragments and produces single-locus binding estimates, aggregate motif footprints, and informative visualizations to support the high-resolution mapping capability of CUT&RUN.
- [CUT&RUNTools](https://bitbucket.org/qzhudfci/cutruntools/src/master/): [a flexible pipeline for CUT&RUN processing and footprint analysis](https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1802-4). CUT&RUNTools is a flexible and general pipeline for facilitating the identification of chromatin-associated protein binding and genomic footprinting analysis from antibody-targeted CUT&RUN primary cleavage data. CUT&RUNTools extracts endonuclease cut site information from sequences of short-read fragments and produces single-locus binding estimates, aggregate motif footprints, and informative visualizations to support the high-resolution mapping capability of CUT&RUN.

- [CUT&RUNTools 2.0](https://github.com/fl-yu/CUT-RUNTools-2.0): [a pipeline for single-cell and bulk-level CUT&RUN and CUT&Tag data analysis](https://academic.oup.com/bioinformatics/article/38/1/252/6318389?login=true). CUT&RUNTools 2.0 is a major update of CUT&RUNTools, including a set of new features specially designed for CUT&RUN and CUT&Tag experiments. Both the bulk and single-cell data can be processed, analyzed, and interpreted using CUT&RUNTools 2.0.

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