Barcoded Polycistronic sgRNA Vector Construction

A single sgRNA lentiviral vector harboring an hU6-sgRNA cassette and an SFFV-mNeonGreen-P2A-ZsGreen1 (referred to as GFP thereafter) cassette was constructed by a standard molecular cloning technique. Annealed sgRNA oligos were inserted into the vector digested by BsmBI. Polycistronic vectors were generated by inserting bU6-sgRNA and mU6-sgRNA cassettes derived from pMJ114 (bovine U6) and pMJ179 (mouse U6) (Addgene: 85995 and 85996) into the single sgRNA vector as previously described (30 (link)). A set of 20-nucleotide barcode sequences were then inserted, resulting in a collection of barcoded polycistronic sgRNA vectors.

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.

Tao Z., Cui Y., Xu X, & Han T. (2022). FGFR redundancy limits the efficacy of FGFR4-selective inhibitors in hepatocellular carcinoma. Proceedings of the National Academy of Sciences of the United States of America, 119(40), e2208844119.

Publication 2022

pMJ114 pMJ179

Bovine Mouse Nucleotide sequences Oligos Sffv Vectors

Corresponding Organization :

Other organizations : Chinese Academy of Medical Sciences & Peking Union Medical College, National Institute of Biological Sciences, Beijing, Beijing Normal University, Tsinghua University

Top 5 similar protocols

Variable analysis

independent variables

Insertion of annealed sgRNA oligos into the vector digested by BsmBI
Insertion of bU6-sgRNA and mU6-sgRNA cassettes derived from pMJ114 (bovine U6) and pMJ179 (mouse U6) into the single sgRNA vector
Insertion of a set of 20-nucleotide barcode sequences into the polycistronic sgRNA vectors

dependent variables

Expression of mNeonGreen-P2A-ZsGreen1 (GFP) cassette

control variables

Construction of the single sgRNA lentiviral vector harboring the hU6-sgRNA cassette and the SFFV-mNeonGreen-P2A-ZsGreen1 (GFP) cassette

positive controls

Not specified

negative controls

Not specified

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.

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?

Revolutionizing how scientists
search and build protocols!