Data · dataset · 2018
Kinetics and fidelity of the repair of Cas9-induced double-strand DNA breaks. Brinkman, E.K. et al
Listed in Teesside University Research Data Repository
Figure 1: We established a clonal K562 cell line with a stably integrated construct that encodes a tightly controlled inducible Cas9 nuclease.
Description
Western blot analysis of Cas9 at various time points after induction of Cas9 by Shield-1. It shows that Cas9 accumulates gradually after induction.
Figure 3, 5, S3, S6: We established a variant of the ligation-mediated PCR assay for the quantification of DNA breaks at a defined location (Chailleux et al., 2014; Dai et al., 2000; Garrity and Wold, 1992). For the detection of broken DNA, we first denature the DNA and subject it to a primer extension reaction using a primer near the break site. This ensures that all cleavage sites are converted into blunt ends, even if resection of the broken ends has occurred.
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Next, an adaptor is ligated to the blunted DNA end, followed by PCR with one primer near the break site and a second primer that is complementary to the adaptor sequence. When analyzed on agarose gels, the samples from cells treated with sgRNA and Shield-1 yielded a band of the expected size. Figure 6: in vitro digestion by CRISPR.
By PCR we first produced double-stranded DNA fragments of 600 – 1,000 bp of 3 loci in the genome. We then incubated each fragment in vitro with the respective Cas9/sgRNA complex to induce DSBs, and investigated the reaction products by agarose electrophoresis. Some samples Cas9 was subsequently denatured by two different heat treatment protocols (20 min 80 ˚C or 5 min 96 ˚C).
DNA was incubated for either 2 hours or overnight.
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Where it is published
- DOI doi.org/10.17632/wg4ssg7pfw.1 ↗
DOI / persistent id · from researchdata tees ac uk
Catalogue records · 1
- OAI-PMH record data.mendeley.com/oai?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Adata… ↗
metadata API · from researchdata tees ac uk
Topics
- From keywords
- Earth & Environmental Science · Genetics · Life Sciences
Provenance · 1 source records, 8 field assertions
| Source | Key | Last seen | Raw |
|---|---|---|---|
| Teesside University Research Data Repository | oai:data.mendeley.com/wg4ssg7pfw.1 | 24 h ago | JSON v1 |
| Field | Assertion | Extractor | Evidence |
|---|---|---|---|
| access_level | source · researchdata tees ac uk | connector:researchdata_tees_ac_uk@1.0.0 | |
| concepts[field].anzsrc:group:3105 | mapping · researchdata tees ac uk | vocabulary-mapper@1.0.0 | keywords['Genetics'] |
| concepts[field].local:field:earth-environmental | mapping · researchdata tees ac uk | connector:researchdata_tees_ac_uk@1.0.0 | |
| concepts[field].local:field:life-sciences | mapping · researchdata tees ac uk | connector:researchdata_tees_ac_uk@1.0.0 | |
| description | source · researchdata tees ac uk | connector:researchdata_tees_ac_uk@1.0.0 | /metadata/dc/description |
| license | source · researchdata tees ac uk | connector:researchdata_tees_ac_uk@1.0.0 | /metadata/dc/rights |
| publication_date | source · researchdata tees ac uk | connector:researchdata_tees_ac_uk@1.0.0 | |
| title | source · researchdata tees ac uk | connector:researchdata_tees_ac_uk@1.0.0 | /metadata/dc/title |