Profiles RNA-binding protein targets without antibody or UV crosslinking using STAMP (APOBEC1-RBP fusion, C-to-U editing), scSTAMP (single-cell), TRIBE/HyperTRIBE (ADAR-RBP, A-to-I editing), DART-seq (APOBEC1-YTH for m6A), or Bullseye/SAILOR edit-site detection pipelines. Use when antibody is unavailable or specificity is doubtful, when single-cell RBP profiling is needed (scSTAMP), or when in vivo RBP profiling without UV is preferred.
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Profiles RNA-binding protein targets without antibody or UV crosslinking using STAMP (APOBEC1-RBP fusion, C-to-U editing), scSTAMP (single-cell), TRIBE/HyperTRIBE (ADAR-RBP, A-to-I editing), DART-seq (APOBEC1-YTH for m6A), or Bullseye/SAILOR edit-site detection pipelines. Use when antibody is unavailable or specificity is doubtful, when single-cell RBP profiling is needed (scSTAMP), or when in vivo RBP profiling without UV is preferred.
Before using code patterns, verify installed versions match. If versions differ:
Python: pip show <package> then help(module.function) to check signatures
CLI: <tool> --version then <tool> --help to confirm flags
If code throws unexpected errors, introspect the installed package and adapt the example to match the actual CLI rather than retrying.
STAMP / Antibody-Free RBP Profiling
"Profile RBP-RNA targets without UV crosslinking or immunoprecipitation" -> Express a fusion of the RBP-of-interest with a deaminase (APOBEC1 for STAMP, ADAR for TRIBE) in cells; the deaminase edits RNA nucleotides adjacent to where the RBP binds, producing a C-to-U (STAMP) or A-to-I (TRIBE, read as A-to-G) editing signature in standard RNA-seq. The targets are recovered computationally from the editing pattern. Three properties make this approach valuable: (a) no UV crosslinking required (works in tissue/in vivo); (b) no IP step (no antibody needed - the RBP itself targets the deaminase); (c) compatible with single-cell readout because the editing signal exists in standard scRNA-seq (scSTAMP, scTRIBE). Trade-off: editing is offset from the binding site (typically 0-50 nt away); resolution is approximate; off-target editing from deaminase alone must be subtracted.
CLI (STAMP, bulk): standard RNA-seq pipeline + Bullseye or SAILOR for C-to-U edit detection vs APOBEC1-only control
CLI (TRIBE, bulk): standard RNA-seq + REDItools2 or JACUSA2 for A-to-I edit detection vs ADAR-only control
CLI (DART-seq for m6A): same as STAMP, with APOBEC1-YTH fusion (YTH is the m6A reader)
Python (scSTAMP single-cell): 10x Genomics or Smart-seq2 pipeline + custom editing-rate quantification per cell + per-cell binding-target inference
CLI (general edit-site detection): JACUSA2 call-2 -r ref.fa -p 8 -F 1024 -A,B treated.bam,control.bam -t pileup.tsv then filter for C-to-U or A-to-I
STAMP (Brannan 2021) is the canonical antibody-free RBP profiling method. TRIBE (McMahon 2016) and HyperTRIBE (Xu 2018) are earlier ADAR-based variants. DART-seq (Meyer 2019) is the m6A-specific application using YTH-fused APOBEC1. scSTAMP (single-cell readout) is part of the same Brannan 2021 method.
Methods Taxonomy
Method
Deaminase
Edit signature
Cells supported
Single-cell
Strength
Fails when
STAMP (Brannan 2021)
APOBEC1
C->U in mRNA (reads as C->T)
Any
Yes (scSTAMP)
Antibody-free; no UV; in vivo
APOBEC1 also edits ssDNA off-target; saturated edits at high APOBEC1 expression
scSTAMP (Brannan 2021)
APOBEC1
C->U per cell
Single cell (10x or Smart-seq2)
Yes (native)
Per-cell RBP profiling
Coverage per cell limits sensitivity; ~25% of cytosines accessible per transcript
TRIBE (McMahon 2016)
ADAR catalytic domain
A->I (reads as A->G in cDNA)
Drosophila standard; mammalian works
Yes (scTRIBE)
First antibody-free
Edits restricted to certain ADAR consensus; lower edit rate than HyperTRIBE
HyperTRIBE (Xu 2018)
ADAR E488Q hyperactive mutant
A->I in much wider context
Drosophila / mammalian
Yes
Higher edit rate than original TRIBE
Hyperactive may edit off-target; needs ADAR-only control
DART-seq (Meyer 2019)
APOBEC1-YTH
C->U near m6A
Any
Yes (scDART)
m6A reader profiling
Indirect (edits near m6A, not at RBP binding sites)
Bullseye
NA (analysis tool)
NA
Any
Yes
STAMP / DART analysis
Just an analysis pipeline
SAILOR
NA (analysis tool)
NA
Any
Yes
RNA editing analysis
Just an analysis pipeline
REDItools2
NA (analysis tool)
NA
Any
NA
Generic RNA editing
Generic; not RBP-specific
JACUSA2 (Piechotta 2022 Genome Biol 23:115)
NA (analysis tool)
NA
Any
NA
Multi-sample edit-site detection
Generic; not RBP-specific
ADAR-CLIP
NA - this is regular ADAR CLIP
NA
NA
NA
CLIP for ADAR
Not an antibody-free method; just a different CLIP target
Methodology evolves; the Brannan lab / Yeo lab 2021 paper is canonical. Verify deaminase fusion expression level (low expression for specificity; saturation degrades specificity).
STAMP vs m6A-Specific Methods
For m6A profiling specifically, antibody-free choices include:
DART-seq (APOBEC1-YTH fusion): This skill covers the methodology, but for m6A detection see clip-seq/m6a-clip. Only ~44% of DART edits fall within DRACH motifs (Guo 2025); strong off-target component.
GLORI (Liu 2023): Antibody-free, chemical, stoichiometric single-base m6A; this is the new (2023) gold standard for m6A. See clip-seq/m6a-clip.
m6Anet (Hendra 2022): Nanopore direct RNA m6A; high AUC on HEK293T.
If the use case is m6A profiling, the m6a-clip skill is the canonical reference; this skill (stamp-antibody-free) focuses on the broader RBP-editing-fusion paradigm where the target is not m6A but the RBP's RNA targets.
Critical Choice: STAMP (APOBEC1) vs TRIBE (ADAR)
Property
STAMP
TRIBE
Deaminase
APOBEC1 (cytidine -> uridine)
ADAR (adenosine -> inosine)
Edit signature
C->U (reads as C->T)
A->I (reads as A->G)
ssRNA preference
Yes (APOBEC1 acts on ssRNA + ssDNA)
No (ADAR acts on dsRNA stems by default; ADAR2cd in TRIBE relaxes this)
Edit clusters per target
10-1000
~5-50 (lower; HyperTRIBE higher)
Off-target
APOBEC1 alone has detectable C->U on ssDNA + RNA
ADAR has weak intrinsic A->I
Spatial offset from RBP binding
0-50 nt
0-30 nt
Cell line tested
HEK293, K562, mouse tissue
Drosophila (original), mouse, human
Single-cell
scSTAMP (Brannan 2021)
scTRIBE
Compatible methods
C->U is rare in mRNA; signal is clean
A->I is common at ALU repeats; baseline ADAR editing competes
Cytosine accessibility
~25-35% of mRNA bases are C; APOBEC1 needs ssRNA
All A residues are potential ADAR targets
Both work; STAMP has more clusters per target (advantage for low-coverage scenarios) and cleaner background (C->U is rare in mRNA). TRIBE has more flexibility (ADAR variants tunable) and lower off-target. Practical choice often comes down to lab familiarity.
scSTAMP / scTRIBE Single-Cell Workflow
The defining advantage of antibody-free RBP profiling is compatibility with single-cell readout. scSTAMP processes 10x Genomics or Smart-seq2 libraries.
Per-cell edit-rate matrix can be integrated with standard scRNA-seq clustering. The per-cell binding profile is reconstructed from cells with sufficient coverage (>= 10000 unique reads typically).
Editing-Site Detection Pipelines
Goal: Recover specific (not background) RBP-fusion-induced editing sites from RNA-seq libraries by subtracting the deaminase-only control.
Approach: Process fusion-sample BAM and deaminase-only-control BAM in parallel; use Bullseye (STAMP/DART), SAILOR (Yeo), or JACUSA2 (general) to call C-to-U (STAMP/DART) or A-to-I (TRIBE/HyperTRIBE) edit sites at edit rate >= 0.1 and coverage >= 10, requiring fusion-vs-control edit ratio > 3 as the specificity threshold.
Bullseye (Meyer lab DART-seq pipeline) is a multi-script Perl pipeline (parseBAM.pl, summarize_sites.pl, find_edit_site.pl) rather than a single binary -- the conceptual flow is shown below; consult the Bullseye repo for the exact per-script invocations.
SAILOR (Yeo lab) is a Snakemake-based pipeline, not a single CLI binary -- launch via the SAILOR Snakefile after editing the config (config.yaml) for input BAMs, background BAM, and reference FASTA.
# SAILOR -- conceptual; SAILOR ships as a Snakemake workflow.# Configure inputs in the SAILOR Snakemake config.yaml, then run:
snakemake -s SAILOR.smk --configfile config.yaml --cores 8
JACUSA2 is a general-purpose RNA editing pipeline, distributed as a Java jar.
Mechanism: At high APOBEC1 expression, the deaminase saturates editing - every accessible C in mRNA is edited, regardless of RBP binding.
Symptom: Edit count per gene >> expected; non-specific editing across mRNAs; APOBEC1-only control has nearly as many edits as the fusion.
Fix: Titrate fusion expression with inducible promoter; aim for low-to-moderate expression giving clean fusion-specific edits. Yeo lab convention: doxycycline-inducible with mid-range dox dose. Compare edits in fusion vs APOBEC1-only; require fusion edits / APOBEC1-only edits > 3.
STAMP -- APOBEC1 off-target on ssDNA
Trigger: APOBEC1 expressed in DNA-replicating cells.
Mechanism: APOBEC1 has intrinsic ssDNA editing activity; some "C->U" calls are actually genomic ssDNA edits read through transcription.
Symptom: Edits cluster at replication-fork regions or LINE-1 elements; non-specific genome-wide.
Fix: Bullseye filters genomic SNVs vs RNA edits via strand information; verify mismatch is C->U on the transcribed strand, not the genomic C->T.
TRIBE -- Editing at ALU repeats
Trigger: TRIBE in mammalian cells; many edits at ALU sequences.
Mechanism: ADAR has baseline activity at ALU dsRNA structures; this is NOT TRIBE-specific signal. ALU edits dominate the apparent target list.
Symptom: Top edited regions are all ALU repeats; target list looks generic.
Fix: Subtract ADAR-only control or wild-type ADAR baseline; filter out ALU-overlapping edits unless RBP is known to bind repeats.
DART-seq -- Spatial offset from m6A
Trigger: DART-seq applied with expectation of single-base m6A resolution.
Mechanism: APOBEC1-YTH edits Cs 0-50 nt from the YTH-bound m6A site. The exact m6A position is not the edit position.
Symptom: DART edits scattered around DRACH motifs; only ~44% of edits within DRACH.
Fix: Treat DART edits as "near m6A"; cross-reference with single-base m6A methods (GLORI, miCLIP2). DART is hypothesis-generating, not precise localization.
scSTAMP -- Coverage limitation per cell
Trigger: scSTAMP on 10x library; per-cell coverage limits target detection.
Mechanism: Single cells have ~5000-50000 mRNA molecules; editing-rate quantification at any single position needs >= 10 reads. Most positions have 0-3 reads per cell.
Symptom: Per-cell binding-target list is sparse; many cells have 0 detected targets.
Fix: Aggregate cells into pseudo-bulk by cluster/cell-type; quantify editing at pseudobulk level; or use ultra-deep Smart-seq2 (~1M reads/cell) instead of 10x for higher per-cell coverage.
No control subtraction
Trigger: STAMP/DART/TRIBE run without deaminase-only control.
Mechanism: Deaminases have intrinsic baseline editing (APOBEC1 ~3-5% C->U; ADAR ~5-10% A->I at ALUs). Without control, all edits look like signal.
Symptom: Edit count enormous; target list non-specific.
Fix: Always run deaminase-only (APOBEC1 or ADAR catalytic domain) control in parallel. Bullseye / SAILOR / JACUSA all support control subtraction.
Strand-specific edit interpretation
Trigger: Generic variant caller used instead of edit-aware tool.
Mechanism: Variant callers report any C->T mismatch; without strand information it is not possible to distinguish C->U (STAMP signal on transcribed strand) from G->A (the reverse complement of C->T on the genome strand from anti-sense reads).
Symptom: Edit count inflated 2x; signal not stranded.
Fix: Use editing-specific tool (Bullseye, SAILOR, JACUSA2) that respects strand. Or filter for proper strand: C->U on +sense and G->A on -sense.
Decision Tree by Use Case
Scenario
Method
Why
Antibody for RBP doesn't exist
STAMP or TRIBE
The original use case
RBP in tissue / in vivo (no UV possible)
STAMP / TRIBE
No CL needed
Single-cell RBP profiling
scSTAMP or scTRIBE
The only practical option
m6A reader profiling (YTHDF)
DART-seq (APOBEC1-YTH)
Specific reader fusion
Drosophila RBP
TRIBE (original development)
Most validated in Drosophila
Mammalian RBP
STAMP (more validated in mammalian)
Yeo lab benchmarks
Need precise binding site
Use CLIP / eCLIP not STAMP/TRIBE
Editing is offset from binding
Repeat-binding RBP
CLIP + CLAM, not TRIBE
ADAR baseline at ALUs swamps TRIBE
Comparison across methods
STAMP + classic eCLIP both
Triangulation increases confidence
Low input cell numbers (< 50k)
scSTAMP
Compatible with sparse libraries
Time-course binding dynamics
STAMP with inducible expression
Live-cell editing accumulates
Cross-link sensitive RBP
STAMP / TRIBE (no UV)
Some RBPs degrade with UV
Reconciliation: STAMP vs CLIP
Pattern
Likely cause
Action
STAMP finds targets eCLIP missed
Targets that crosslink poorly; or low-abundance
Validate orthogonally; STAMP often more sensitive for low-abundance targets
eCLIP finds targets STAMP missed
RBP-RNA contact too far from accessible C; or APOBEC1 saturated
Titrate fusion expression; verify APOBEC1-only control
TRIBE editing dominated by ALUs
ADAR baseline activity
Subtract ADAR-only; filter ALU repeats
Discordant target lists across labs for same RBP
Fusion expression varies; control differs
Standardize protocols; cross-validate
scSTAMP pseudobulk = bulk STAMP
Cell aggregation correct
Trust both for low-coverage targets
scSTAMP per-cell sparse
Coverage limitation
Pseudobulk by cluster; or use Smart-seq2
Operational rule: STAMP/TRIBE for hypothesis-generation, antibody-free profiling, or single-cell. CLIP/eCLIP for high-resolution validation. Best paper figure: STAMP + eCLIP concordance for top targets.
Guo W et al 2025 Mol Cell 85:1233 (DART-seq DRACH reanalysis, 44% within DRACH)
(SAILOR: pipeline by Yeo lab; documented at github.com/YeoLab/SAILOR -- specific peer-reviewed citation has not been confirmed; consult current literature.)
Piechotta M et al 2017 BMC Bioinformatics 18:7 (JACUSA1).
Piechotta M et al 2022 Genome Biol 23:115 (JACUSA2 -- the multi-sample call-2 mode used above).
Picardi E & Pesole G 2013 Bioinformatics 29:1813 (REDItools)
Tegowski M et al 2022 Mol Cell 82:868 (scDART single-cell DART-seq)
Related Skills
clip-seq/m6a-clip - DART-seq is part of the m6A toolkit
