Collection Citation: A. Felicia Adebanjo, Madeline Walsh, Jessica Simon, Raining Wang, Melinda Wheelock, Allyssa Vandi, Aman Shihora, Sriram Pendyala, Dan Holmes, Abigail McGee, Melissa Hopkins, Jordan Opsahl, Dustin J. Maly, Douglas M. Fowler 2025. LABEL-seq Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2141xg1y/v1
License: This is an open access collection distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this collection
Created: July 22, 2025
Last Modified: October 24, 2025
Collection Integer ID: 223319
Keywords: PCR amplification, SapI enzyme, Barcode sequence design, Electro-competent cells, Library gene, Bottlenecking, Passage cells, RPLP cell lines, Homogenization, Reverse Transcription, Amplicon, Transduction, Lentivirus, ERK inhibitor, step cloning system compatible with golden gate assembly, nucleotide barcode, small ms2 rna hairpin, ms2 phage coat protein, barcode sequence, cloning of the goi variant library, step cloning system, golden gate assembly, protein variant, barcode sequences to the vector, specific barcode, pair of sapi recognition sequence, stable circular rna, cloning, expression of stable circular rna, seq v2, quantitative measurement of gene activity, circrna architecture, ms2 hairpin, sapi recognition sequence, gene, barcode, enrichment for biochemical analysis, gene activity, barcoded vector, seq protocol pre, protein abundance
Funders Acknowledgements:
NHGRI IGVF
Grant ID: 1UM1HG011969-01
Abstract
Pre-barcoded Vector Design:
This protocol outlines the materials and methods for conducting a LABEL-seq (Labeling with Barcodes and Enrichment for Biochemical Analysis by Sequencing) assay. The assay utilizes designated plasmids containing Bxb1-attB sites and MS2 phage coat protein (MCP) binding elements that link with small MS2 RNA hairpins. A gene of interest (GOI) can be fused to MCP either at the N-terminus (N-term) or C-terminus (C-term), enabling the expression of stable circular RNAs (circRNAs) that incorporate MS2 hairpins along with a unique 18-nucleotide barcode. Each protein variant is tagged with a specific barcode, allowing quantitative measurement of gene activity and protein abundance. The current (as of 2025) working system (LABEL-seq v2) is modified based on the published version (Simon et al. 2024). There are two major modifications made for a two-step cloning system compatible with Golden Gate Assembly. First, a pair of BsmBi/ Esp3I recognition sequences, located on the circRNA architecture, is used to add barcode sequences to the vector. Secondly, a pair of SapI recognition sequences were added to assist cloning of the GOI variant library.
Guidelines
Barcodes and GOI Design
Barcode sequence design:
Below is the final amplicon sequence for adding barcodes to either N-& C- terminal plasmids.
The template sequence is a double-stranded DNA fragment with a 18nt bases (N) barcode.The
lowercase indicates fragment-specific overhangs for backbone assembly using BsmBi/EspsI enzyme.
This double-stranded DNA is created by a Klenow fill-in process from a single stranded template with
primer 5’-ATCAATCCCCTACACCTTCGCG-3’
Gene of Interest flanking design:
LABEL-seq v2 vectors have two pre-existing SapI sites and pre-selected fragment specific overhangs
ready to receive GOI sequence with complimentary flasking ends. The specific flasking sequences are as
below:
In the N-terminus system, 5’-atg and 5’-CTA (reverse complement of 5’-tag”) are the pre-selected
fragment-specific overhangs in the backbone. The insert fragment must contain the SapI recognition
sequences (in red) and overhangs (indicated as lowercase) as the following:
In the C-terminus system: 5’-atg and 5’-CGT (reverse complement of 5’-acg) are the pre-selected
fragment-specific overhangs in the backbone to receive a compatible insert fragment. The insert fragment
must contain the SapI recognition sequences (in red) and overhangs (indicated as lowercase) as the
following:
*A stop codon should not be placed at the end of GOI sequence when assembling to the C-terminus
backbone. It is already placed at the end of the MCP. Albeit, in the N-terminus system, the stop codon is
necessary for the GOI insert. It is a part of the SapI overhang.
Both Start and Stop codons cannot be mutated.
Wild-type and destabilizing variants design:
Wild-type of Gene of Interest:
Wild type sequences for gene of interest(s) can be obtained from PCR amplification of existing template,
or synthesized as double-stranded DNA fragments. In both cases, the interior sequence should be free of
SapI recognition sequences and flanked by the above sequences suitable for the desired designation
vectors.
Variant Library design:
A protein variant library can be designed and synthesized as a site-saturation mutagenesis library,
introducing 22 possible amino acid substitutions (20 common codons along with one deletion and Stop)
and at each codon position, excluding the start and stop codons of a gene.
