Feb 25, 2026

Variant Abundance Assay V.2

DOI
https://dx.doi.org/10.17504/protocols.io.dm6gpmwm1gzp/v2
  • Madeline Walsh1,
  • A. Felicia Adebanjo1,
  • Jessica Simon1,
  • Melissa Hopkins1,
  • Raining Wang1,
  • Melinda Wheelock1,
  • Allyssa Vandi1,
  • Dan Holmes1,
  • Aman Shihora1,
  • Sriram Pendyala1,
  • Douglas M. Fowler1,
  • Dustin J. Maly1
  • 1University of Washington
Melinda K. Wheelock
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DOI: https://dx.doi.org/10.17504/protocols.io.dm6gpmwm1gzp/v2
Protocol Citation: Madeline Walsh, A. Felicia Adebanjo, Jessica Simon, Melissa Hopkins, Raining Wang, Melinda Wheelock, Allyssa Vandi, Dan Holmes, Aman Shihora, Sriram Pendyala, Douglas M. Fowler, Dustin J. Maly 2026. Variant Abundance Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpmwm1gzp/v2Version created by Melinda K. Wheelock
License: This is an open access  protocol  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 protocol
Created: February 24, 2026
Last Modified: February 25, 2026
Protocol  Integer ID: 243950
Keywords: Supernatant, Halotag, Lyse cells, abundance assay with flag, description of abundance assay, abundance assay, halotag enrichments this protocol, halotag enrichment, assay, flag, variant abundance assay quantify intracellular abundance, protein variants via ratiometric sequencing, variant abundance assay quantify, tagged protein variant, circrna barcode, intracellular abundance, ratiometric sequencing, pooled tdmcp, immunoprecipitation, tdmcp standard, tdmcp
Abstract
Quantify intracellular abundance of pooled tdMCP-tagged protein variants via ratiometric sequencing of MS2-circRNA barcodes co-enriched in parallel Flag (variant) and Myc (tdMCP standard) immunoprecipitations.
Materials
  • Tri-Isolate RNA Pure Kit (IBI Scientific, Catalog # FEREO0384)
  • Halolink resin slurryPromegaCatalog #G1913
  • Eppendorf Protein LoBind tubesEppendorfCatalog #022431064
  • Halolink compatible protease inhibitorPromegaCatalog #G6521
  • Anti-Flag m2 magnetic beadsMerck MilliporeSigma (Sigma-Aldrich)Catalog #M8823
  • Tergitol (NP-40 solution)Merck MilliporeSigma (Sigma-Aldrich)Catalog #NP40S
  • Tris BaseMerck MilliporeSigma (Sigma-Aldrich)Catalog #252859
  • GlycerolMerck MilliporeSigma (Sigma-Aldrich)Catalog #G5516
  • Magnesium chloride hexahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #442611
  • Sodium fluorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S7920
  • Phenylmethanesulfonyl fluorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #P7626 SIGMA
  • RiboLock RNase Inhibitor (40 U/µL)Thermo FisherCatalog #EO0382
  • Sodium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9625
  • Doxycycline hyclateMerck MilliporeSigma (Sigma-Aldrich)Catalog #D5207
  • DMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #472301
  • SCH772984MedChemExpressCatalog #HY-50846
  • DMEM, high glucoseThermo FisherCatalog #11965118
  • Fetal Bovine Serum, Tet system approved, USDA-approved regionsThermo Fisher ScientificCatalog #A4736401
  • Penicillin-Streptomycin (10,000 U/mL)Thermo Fisher ScientificCatalog #15140122

Cells & Plasmids
  • HEK293 RPLP cells with recombined tdMCP-variant library
  • pLJM1 2xMyc-tdMCP-SNAP (standard)
  • Packaging plasmids: pMDLg/pRRE-Gag and Pol (Addgene #12251), pRSV-Rev (Addgene #12253), pVSV-G (Addgene #138479)
  • Dummy RNA (ordered from IDT)

Transfection and Cell Culture
  • Fugene HD (Promega E2311)
  • Trypsin
  • Doxycycline (Sigma-Aldrich D9891-5G) (2 µg/mL)
  • SCH772984 (Selleckchem S7101) (100 nM)

Lysis and Inhibitors
  • Halt Protease Inhibitor Cocktail (Thermo 78429)
  • PMSF (Sigma P7626)
  • RiboLock RNase Inhibitor (Thermo EO0381)
  • NP-40 (Igepal CA-630, Sigma I8896)

Beads & Antibodies

  • Anti-Flag M2 Magnetic Beads (Sigma M8823)
  • Anti-Myc Magnetic Beads (Thermo 88842)

RNA Extraction & Cleanup

  • TRI-Isolate RNA Pure Kit (IBI Scientific IBI-32110)
  • Zymo RNA Clean & Concentrator-5 Kit (Zymo R1015)

Reverse Transcription & PCR

  • SuperScript IV Reverse Transcriptase (Thermo 18090010)
  • Q5 High-Fidelity Master Mix (NEB M0494)
  • AMPure XP beads (Beckman Coulter A63880)
  • Custom primers (JJS1, JJS8–JJS28) from IDT

Sequencing

  • Illumina NextSeq P3 50 Kit (varies by run type)
  • Genewiz Amplicon-EZ service (no catalog number)


Equipment

  • Serological pipettor
  • Micropipettes and tips
  • Centrifuge
  • Magnetic bead rack
  • End-over-end rotator in 4C

Buffer Recipes


Final concentrations of buffers:

  • Resuspension buffer

○ 50 millimolar (mM) Tris pH 7.6 ○ 150 millimolar (mM) NaCl ○ 5% glycerol ○ 4 millimolar (mM) MgCl2 ○ 10 millimolar (mM) NaF ○ 2 millimolar (mM) PMSF

  • 1x Promega protease inhibitor

○ 4 uL/mL Ribolock RNase inhibitor ○ 300 nanomolar (nM) dummy RNA

  • Lysis buffer

○ 50 millimolar (mM) Tris pH 7.6 ○ 150 millimolar (mM) NaCl ○ 5% glycerol ○ 4 millimolar (mM) MgCl2 ○ 10 millimolar (mM) NaF ○ 2 millimolar (mM) PMSF ○ For abundance assay:
  • 1x Promega protease inhibitor

○ 4 uL/mL Ribolock RNase inhibitor ○ 0.2% NP40

  • Wash buffer

○ 50 millimolar (mM) Tris pH 7.6 ○ 150 millimolar (mM) NaCl ○ 5% glycerol ○ 4 millimolar (mM) MgCl2 ○ 10 millimolar (mM) NaF ○ 2 millimolar (mM) PMSF ○ 0.1% NP40

Stock buffers/reagents (prepare in advance)


5x RIPA (200mL)

AB
Tris6.057g
NaCl8.677g
Glycerol50mL
Magnesium chloride hexahydrate813mg
MillQ water200mL
Adjust pH to 7.6.


Note
Make stock and aliquot, storing at -20C

1M PMSF stock (1mL)

AB
PMSF174mg
DMSO1mL
Sonicate to dissolve.


Buffers to make day of assay

Note
Ultimately, you only need the resuspension, lysis, and wash buffers which can each be made individually. For easier preparation, the following describes how to make each buffer from Buffer A (1x modRIPA without inhibitors) and Buffer B (1x modRIPA with inhibitors). Buffer A and Buffer B are used only to make resuspension, lysis, and wash buffers.

Buffer A - 1x modRIPA (10mL)

AB
5x RIPA2mL
MilliQ water8mL
NaF4.2mg
1M PMSF stock (3.48mg PMSF)20μL
Sonicate to dissolve (can heat in water bath as needed, <5min). PMSF may crash out after adding to buffer, so make sure to fully redissolve.
Buffer B - modRIPA with inhibitors (10mL)

  • 10 mL Buffer A
  • 40 µL Ribolock RNase inhibitor
  • For abundance assay:
○ 200 µL 50x Promega protease inhibitor
  • For activity assay:
○ 1 mini Halt protease inhibitor tablet ○ 100 µL phosphatase inhibitor cocktail 2 ○ 100 µL phosphatase inhibitor cocktail 3
The mini Halt protease inhibitor tablet is the most difficult reagent to fully dissolve in solution, so add to Buffer A before adding anything else and sonicate to dissolve (can heat in water bath as needed, <5min).

Resuspension Buffer (1mL)

  • 1 mL Buffer B
  • 30 µL 10 micromolar (µM) dummy RNA

Lysis Buffer (1mL)

AB
Buffer B1mL
NP402μL
Sonicate to dissolve NP40 in solution. NP40 is a detergent so avoid vortexing.


Wash Buffer (1mL)

AB
Buffer A1mL
NP401μL
Sonicate to dissolve NP40 in solution. NP40 is a detergent so avoid vortexing.

Troubleshooting
Problem
Low overall barcode reads
Solution
ensure sufficient input cells, verify RT efficiency (JJS1 priming), confirm PCR conditions and no inhibition from carryover TRIzol.
Problem
Apparent barcode exchange / high mis-association
Solution
make sure dummy RNA concentration is correct (400 ng/µL in resuspension buffer for +dummy), keep samples cold, minimize time between lysis and bead capture.
Problem
High background in IPs
Solution
increase wash stringency (extra washes, higher salt in modRIPA), check bead quality and antibody activity.
Problem
Low RNA yield from beads
Solution
minimize bead drying, use carrier (glycogen) in precipitation if needed, concentrate RNA via Zymo columns.
Before start
NOTE: Protocol begins after cells are recombined with library and selected
Determine the number of cells needed for the experiment ● Aim for ~240 cells/barcode per replicate ○ Allocate 60 cells/barcode per replicate for Flag enrichment ○ Allocate 180 cells/barcode per replicate for HaloTag enrichment
Example: For a 200k barcode library, you need 48M cells (2.5 x 15cm plates) per replicate
Day −2 to −1: Plate & transfect Myc-tdMCP standard
Plate Flag-tdMCP-BRaf library cells into 15-cm dishes at ~45% confluency the day before transfection.

  1. For each 15-cm dish, transiently transfect cells by combining the following and incubating at room temperature for 15 min. Add mixture dropwise to cells.
17 µg pCDNA5 Myc-tdMCP-SNAPtag
1.7 µg pMAX-GFP
70 µL Fugene HD
2 mL serum-free DMEM
Incubate 48 h after transfection.
Add 2 µg/mL doxycycline
Maintain cells in 100 nM SCH772984 if using cells that drive elevated levels of pErk. Wash out inhibitor 24 hrs prior to cell lysis.
1 day before enrichment
Plate Flag-tdMCP-BRaf library cells into 15-cm dishes at ~45% confluency the day before 77transfection298.
Harvest and allocate cells
10m
Pool plates and harvest cells by trypsinization.
Count and aliquot cells into the following per replicate:

  • 2 × no-dummy samples: 30 × 10⁶ cells each.
  • 2 × +dummy samples: 10 × 10⁶ cells each.
  • 1 × input sample: 10 × 10⁶ cells.
Resuspend cells
1h 40m
Resuspend each aliquot in resuspension buffer (no detergent):

  • no-dummy samples → 1,650 µL resuspension buffer.
  • +dummy and input samples → 550 µL resuspension buffer. For +dummy samples include dummy total RNA to a final concentration of 400 ng/µL. (Dummy RNA reduces barcode mis-association.)
Lyse cells
Immediately add an equal volume of cold modRIPA (0.1% NP-40) to each resuspension (i.e., add 1,650 µL to each no-dummy tube, 550 µL to each +dummy/input tube). Addition of detergent marks the start of lysis incubation. Keep tubes on ice.
Clear lysates by centrifugation at 17,000 × g for 10 min at 4°C. Transfer clarified supernatant to low-bind tubes.
For downstream enrichment split cleared lysates into aliquots matched to bead volumes used below:
no-dummy cleared lysates → split into 1.5 mL aliquots.
+dummy cleared lysates → split into 0.5 mL aliquots.
Immunoprecipitation
Prepare beads at 4°C and equilibrate in modRIPA
Add bead slurry to each aliquot immediately and rotate for 2 hours at 4°C:
no-dummy aliquots: 150 µL bead slurry per aliquot (for either Anti-Flag or Anti-Myc capture).
+dummy aliquots: 50 µL bead slurry per aliquot (for either capture).
These bead slurry volumes (150 µL vs 50 µL) match the paper’s no-dummy vs +dummy conditions. Incubate 2 hours rotating at 4°C.
After incubation, collect beads on a magnetic rack and discard supernatant.
Washes and RNA stabilization
Wash beads 3× with 500 µL cold modRIPA (0.1% NP-40) per wash. For each wash, gently invert or flick tubes and then collect beads on magnet. Keep everything cold and RNase-free.
After final wash, remove all residual buffer and resuspend beads in 75 µL nuclease-free water + 300 µL TRIzol. For input sample, resuspend cell pellet in 1.5 mL TRIzol. Store at −20°C if not extracting immediately.
RNA extraction & cleanupUntitled section
Extract RNA according to TRI-Isolate kit instructions.
Clean and concentrate RNA using Zymo RNA Clean & Concentrator kit; elute in ~10 µL nuclease-free water.
Reverse transcription & amplicon library prep
Reverse transcription: use SuperScript IV with gene-specific RT primer JJS1 (TTACGACTCGTCGTCACT) following manufacturer protocol. Use RNase inhibitor throughout setup.
PCR round 1: amplify the circRNA barcode region using the round-1 forward primer (e.g., JJS8) and the appropriate round-1 reverse primer(s) matching your library build (JJS9–JJS15). Use high-fidelity polymerase (Q5). Cycle numbers: test to avoid over-amplification (e.g., 18–22 cycles; choose based on input).
PCR round 2 (adapter/indexing): perform a second PCR to append Illumina adapters and indices using the round-2 forward primers (JJS16–JJS23, choose one per sample) and the round-2 reverse primer JJS24; add index primers JJS27/JJS28 as required. Purify PCR products (AMPure XP) and QC by gel / Bioanalyzer.
Quantify amplicons, pool equimolarly and sequence on Illumina platform (or send to Amplicon-EZ). Provide custom read primers JJS25/JJS26 to the sequencing provider if you are using non-standard read priming.
Sequence on Illumina NextSeq 2000 - use custom barcode read primers if required (JJS25/JJS26).
Data Processing
From sequencing data, obtain the barcode counts for each sample
- Counts_flag (variant capture)
- Counts_myc (tdMCP standard capture)
Use the following formula to compute the per-barcode ratio:
r_barcode = (counts_flag)/(counts_myc)

Compute the average r_barcode for each variant
Normalize to WT
Abundance score = mean_r_variant/r_WT
Protocol references
Simon JJ, Fowler DM, Maly DJ. Multiplexed profiling of intracellular protein abundance, activity, interactions, and druggability with LABEL-seq. Nat Methods. 2024 Nov;21(11):2094-2106. doi: 10.1038/s41592-024-02456-7. Epub 2024 Oct 21. PMID: 39433876; PMCID: PMC11785348.