Huh7 and HepaRG cells were used to introduce indels targeting essential nucleotides of the G-quadruplex structure in 5′UTR region of STAT1 gene.33 (link) Ribonucleoprotein of Cas9–2NLS (10 pmol) and guide RNA (50 pmol, Synthego) were loaded onto a 10 μL Neon Tip and electroporated into 1×105 Huh7 and HepaRG cells, using Neon Transfection System at 1200 V, for 20 ms and four pulses (ThermoFisher Scientific), according to manufacturer’s instructions. Genomic DNA was isolated and used for rapid PAGE genotyping34 (link) to validate incorporation of indels, 48 hours after electroporation. Validated pools of cells were subjected to clonal selection. Isolated single colonies were confirmed by rapid PAGE genotyping and allelic sequencing.
Protocol detail
CRISPR-mediated STAT1 G-quadruplex disruption
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
Allelic
Cells
Clonal
Electroporation
G quadruplex
Gene
Genomic
Indels
Neon
Nucleotides
Pulses
Ribonucleoprotein
Stat1
Transfection
Corresponding Organization :
Other organizations : Purdue University West Lafayette, Centre International de Recherche en Infectiologie, Center for Excellence in Molecular Cell Science, Hôpital de la Croix-Rousse
Top 5 similar protocols
Variable analysis
independent variables
- Introducing indels targeting essential nucleotides of the G-quadruplex structure in 5′UTR region of STAT1 gene
- Cas9–2NLS (10 pmol) and guide RNA (50 pmol, Synthego) loaded onto a 10 μL Neon Tip
- Electroporation of Cas9–2NLS and guide RNA into Huh7 and HepaRG cells using Neon Transfection System at 1200 V, for 20 ms and four pulses
- Incorporation of indels, validated 48 hours after electroporation
- Validated pools of cells subjected to clonal selection
- Isolated single colonies confirmed by rapid PAGE genotyping and allelic sequencing
- Huh7 and HepaRG cell lines used
- Rapid PAGE genotyping method used to validate incorporation of indels
- Electroporation parameters: 1200 V, 20 ms, four pulses
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!