sgRNA FlowCode Plasmids Kit
(Kit #
1000000286
)
Depositing Lab: Adrian Liston
The following kit consists of a set of 56 FlowCode plasmids suited for CRISPR knockdown experiments. Each plasmid expresses a unique triple barcode from the following epitopes: FLAG, S, C, V5, VSVg, HA, Ollas, and AU1. These plasmids are only intended for the expression of sgRNA sequences. sgRNAs can be cloned into the plasmids using BbsI recognition sites. Note the plasmids do not natively encode Cas9.
This kit will be sent as bacterial glycerol stocks in 96-well plate format.
Original Publication
Systematic in vivo testing identifies context- and tissue-dependent migratory requirements for regulatory and conventional T cells. In submission
Description
The FlowCode system is a retroviral barcode platform designed for in vivo multiplexed CRISPR screens with readout by spectral flow cytometry (advanced imaging method that reads the full emission spectra of fluorophores; requires a spectral cytometer). Derived from the Pro-Code system (Brian Brown Lab, Wroblewska et al., 2018 (Link opens in a new window)), FlowCode uses combinatorial triplet arrangements of eight short linear epitopes (FLAG, S, C, V5, VSVg, HA, OLLAS, and AU1) fused to histone H2bc3, enabling stable, long-lived barcode detection at single-cell resolution. Each unique triplet combination serves as a barcode to track a distinct genetic perturbation within a single experimental system.
This kit contains 56 unique retroviral plasmids (FlowCode1–FlowCode56), each encoding a distinct triplet epitope barcode alongside an hU6-driven sgRNA cassette. Plasmids carry an ampicillin resistance marker and use BbsI restriction sites for sgRNA oligo cloning (refer to the accompanying cloning protocol for oligo design and ligation guidance). Retroviral particles produced from these plasmids are used to transduce the target cell population (refer to the transduction protocol for optimised efficiency in T cells and recommendations for other cell types).
Note: these plasmids do not encode Cas9. A Cas9-expressing system must be provided separately for gene knockout to occur.
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 sgRNA FlowCode Plasmids Kit was a gift from Adrian Liston (Addgene kit #1000000286)."
sgRNA FlowCode Plasmids Kit - #1000000286
- 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 |
Inventory
| Well | Plasmid | Resistance |
|---|---|---|
| A / 1 | Flowcode_01 |
|
| A / 2 | Flowcode_02 |
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| A / 3 | Flowcode_03 |
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| A / 4 | Flowcode_04 |
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| A / 5 | Flowcode_05 |
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| A / 6 | Flowcode_06 |
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| A / 7 | Flowcode_07 |
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| A / 8 | Flowcode_08 |
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| A / 9 | Flowcode_09 |
|
| A / 10 | Flowcode_10 |
|
| A / 11 | Flowcode_11 |
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| A / 12 | Flowcode_12 |
|
| B / 1 | Flowcode_13 |
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| B / 2 | Flowcode_14 |
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| B / 3 | Flowcode_15 |
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| B / 4 | Flowcode_16 |
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| B / 5 | Flowcode_17 |
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| B / 6 | Flowcode_18 |
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| B / 7 | Flowcode_19 |
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| B / 8 | Flowcode_20 |
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| B / 9 | Flowcode_21 |
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| B / 10 | Flowcode_22 |
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| B / 11 | Flowcode_23 |
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| B / 12 | Flowcode_24 |
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| C / 1 | Flowcode_25 |
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| C / 2 | Flowcode_26 |
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| C / 3 | Flowcode_27 |
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| C / 4 | Flowcode_28 |
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| C / 5 | Flowcode_29 |
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| C / 6 | Flowcode_30 |
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| C / 7 | Flowcode_31 |
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| C / 8 | Flowcode_32 |
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| C / 9 | Flowcode_33 |
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| C / 10 | Flowcode_34 |
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| C / 11 | Flowcode_35 |
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| C / 12 | Flowcode_36 |
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| D / 1 | Flowcode_37 |
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| D / 2 | Flowcode_38 |
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| D / 3 | Flowcode_39 |
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| D / 4 | Flowcode_40 |
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| D / 5 | Flowcode_41 |
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| D / 6 | Flowcode_42 |
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| D / 7 | Flowcode_43 |
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| D / 8 | Flowcode_44 |
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| D / 9 | Flowcode_45 |
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| D / 10 | Flowcode_46 |
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| D / 11 | Flowcode_47 |
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| D / 12 | Flowcode_48 |
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| E / 1 | Flowcode_49 |
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| E / 2 | Flowcode_50 |
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| E / 3 | Flowcode_51 |
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| E / 4 | Flowcode_52 |
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| E / 5 | Flowcode_53 |
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| E / 6 | Flowcode_54 |
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| E / 7 | Flowcode_55 |
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| E / 8 | Flowcode_56 |
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