Barcoded Self-Reporting Transposon Calling Card Collection
(Kit #
1000000213
)
Depositing Lab: Rob Mitra
'Calling Cards' technology using self-reporting transposons (SRTs) enables the identification of DNA-protein interactions through RNA sequencing. The Mitra lab Barcoded Self-Reporting Transposon Calling Card Collection simplifies bulk calling cards experiments, enables barcoding of experimental conditions, and allows for pooled library preparations that drastically reduce cost and labor.
This collection contains two sets of barcoded SRTs:
- piggyBac self-reporting transposons with puromycin marker for mammalian calling cards experiments in cultured cells
- piggyBac self-reporting transposons with tdTomato marker in an AAV backbone for in vivo experiments
This kit will be sent as bacterial glycerol stocks in 96-well plate format.
Original Publication
Measuring transcription factor binding and gene expression using barcoded self-reporting transposon calling cards and transcriptomes. Lalli M, Yen A, Thopte U, Dong F, Moudgil A, Chen X, Milbrandt J, Dougherty JD, Mitra RD. NAR Genom Bioinform. 2022 Aug 31;4(3):lqac061. doi: 10.1093/nargab/lqac061. eCollection 2022 Sep. PubMed (Link opens in a new window) Article (Link opens in a new window)
Description
This collection of plasmids is a set of barcoded self-reporting transposons used for ‘calling card’ experiments. The calling card assay enables the identification of DNA-protein interactions using RNA-seq. Overexpression of a piggyBac transposase fused to a DNA-binding protein of interest will insert a self-reporting transposon (SRT) into the genome. The SRT consists of a promoter driving a reporter such as a fluorescent protein or puromycin resistance cassette flanked by piggyBac terminal repeats (Figure 1). No polyadenylation signal sequence after the reporter allows transcription through the SRT into the genome. The transcribed RNA can be collected with standard RNA purification techniques and used to map the SRT-genome junction, thereby indicating where the DNA-binding protein binds in the genome. This collection of plasmids adds a barcode in the SRT, proximal to the SRT-genome junction, that can be used to identify distinct binding events in a population of cells. Specific barcode information is found on each individual plasmid page.
This collection contains:
- 23 barcoded piggyBac self-reporting transposons with puromycin marker for mammalian calling cards experiments in cultured cells
- 20 barcoded piggyBac self-reporting transposons with tdTomato marker in an AAV backbone for in vivo experiments
- Figure 1. A puromycin-resistant self-reporting piggyBac transposon after integrating into the genome. The EF1alpha promoter drives expression of the puromycin resistance gene as a reporter. The cassette is flanked by piggyBac terminal repeats (TR), comprising a 19 base pair internal repeat (in blue), a 3 base pair spacer (in black), and a 13 base pair terminal inverted repeat region (in red). Barcodes that are 4 nucleotides in length are inserted within the 13 bp terminal repeat sequence.
Kit-Specific Resources
See protocols.io for the Barcoded Calling Cards and Transcriptomes Library Protocol (Link opens in a new window)and Code Ocean for the processing pipeline (Link opens in a new window).
How to Cite this Kit
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.
For your Materials and Methods section:
“The Barcoded Self-Reporting Transposon Calling Card Collection was a gift from Rob Mitra (Addgene kit #1000000213).”
For your Reference section:
Measuring transcription factor binding and gene expression using barcoded self-reporting transposon calling cards and transcriptomes. Lalli M, Yen A, Thopte U, Dong F, Moudgil A, Chen X, Milbrandt J, Dougherty JD, Mitra RD. NAR Genom Bioinform. 2022 Aug 31;4(3):lqac061. doi: 10.1093/nargab/lqac061. eCollection 2022 Sep. PubMed (Link opens in a new window) Article (Link opens in a new window)
Barcoded self-reporting transposon calling card collection - #1000000213
- Resistance Color Key
Each circle corresponds to a specific antibiotic resistance in the kit plate map wells.
- Inventory
Searchable and sortable table of all plasmids in kit. The Well column lists the plasmid well location in its plate. The Plasmid column links to a plasmid's individual web page.
- Kit Plate Map
96-well plate map for plasmid layout. Hovering over a well reveals the plasmid name, while clicking on a well opens the plasmid page.
Resistance Color Key
| Ampicillin |