REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Mouse monoclonal anti-FLAG (clone M2)	Sigma-Aldrich	Cat#F1804; RRID: AB_262044
Chemicals, peptides, and recombinant proteins
RNAse Away Reagent	Thermo Fisher Scientific	Cat#10328011 Note: For removal of RNAse contamination from surfaces, tools and gloves!
H2O, RNAse and Endotoxin Tested	AmericanBio	Cat#AB02128-00500; CAS: 7732-18-5 Note: For buffer preparation.
EtOH, pure alcohol, 200 proof	AmericanBio	Cat#AB00515-00500; CAS: 64-17-5 Note: For buffer preparation and bead washing steps.
RPMI 1640	Thermo Fisher Scientific	Cat#11875093
Tetracycline-negative FBS	Gemini Bio-Products	Cat#100-800
L-glutamine	Thermo Fisher Scientific	Cat#25030081
Penicillin-Streptomycin	Thermo Fisher Scientific	Cat#15140122; CAS: 3810-74-0
Doxycycline	Sigma-Aldrich	Cat#D3447; CAS: 10592-13-9
Tris HCl, 1 M solution, pH 7.4	AmericanBio	Cat#AB14044-01000; CAS: 7732-18-5, 77-86-1
Sodium chloride, 5 M solution	AmericanBio	Cat#AB13198-01000; CAS: 7732-18-5, 7647-14-5
NP-40 Surfact-Amps™ Detergent Solution	Thermo Fisher Scientific	Cat#85124; CAS: 9016-45-9
SDS, 10% Solution, RNase-free	Thermo Fisher Scientific	Cat#AM9822; CAS: 151-21-3
Sodium deoxycholate	Sigma-Aldrich	Cat#D6750-10G; CAS: 302-95-4
cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail	Sigma-Aldrich	Cat#11836170001; CAS: 25322-68-3, 30827-99-7
SUPERase⋅In™ RNase Inhibitor (20 U/μL)	Thermo Fisher Scientific	Cat#AM2696; CAS: 56-81-5
EDTA, 0.5 M Solution, pH 8.0	AmericanBio	Cat#AB00502-01000; CAS: 7732-18-5, 6381-92-6
Magnesium chloride, 1 M solution	AmericanBio	Cat#AB09006-00100; CAS: 7732-18-5, 7791-18-6
TWEEN 20	AmericanBio	Cat#AB02038-00500; CAS: 9005-64-5
Tris, 2 M solution, pH 7.5	AmericanBio	Cat#AB14116-01000; CAS: 7732-18-5, 77-86-1
Potassium chloride solution	Sigma-Aldrich	Cat#60135; CAS: 7732-18-5, 7447-40-7
Triton X-100 Detergent	Bio-Rad	Cat#1610407; CAS: 9002-93-1
Tris-HCl 1 M, pH 6.5	Teknova	Cat#T1065; CAS: 77-86-1
DTT (5 g)	DOT Scientific	Cat#DSD11000-5; CAS: 3483-12-3
UltraPure™ 1 M Tris-HCI Buffer, pH 7.5	Thermo Fisher Scientific	Cat#15567027
UltraPure™ Urea	Thermo Fisher Scientific	Cat#15505035
NuPAGE™ MOPS SDS Running Buffer (20×)	Thermo Fisher Scientific	Cat#NP0001; CAS: 151-21-3, 68-12-2
NuPAGE™ Transfer Buffer (20×)	Thermo Fisher Scientific	Cat#NP00061
Methanol ACS reagent, ≥99.8%	Sigma-Aldrich	Cat#179337; CAS: 67-56-1
Novex™ TBE Running Buffer (5×)	Thermo Fisher Scientific	Cat#LC6675
Dynabeads™ Protein G for Immunoprecipitation	Thermo Fisher Scientific	Cat#10004D; CAS: 26628-22-8
Ambion™ RNase I (100 U/μL)	Thermo Fisher Scientific	Cat#AM2295; CAS: 56-81-5
TURBO™ DNase (2 U/μL)	Thermo Fisher Scientific	Cat#AM2239; CAS: 56-81-5
FastAP Thermosensitive Alkaline Phosphatase (1 U/μL)	Thermo Fisher Scientific	Cat#EF0651; CAS: 56-81-5, 9002-93-1
AffinityScript Multiple Temperature Reverse Transcriptase	Agilent Technologies	Cat#600107; CAS: 56-81-5, 7447-40-7
T4 Polynucleotide Kinase	New England Biolabs	Cat#M0201L; CAS: 56-81-5, 60-00-4
100% DMSO	New England Biolabs	Cat#B0515AVIAL; CAS: 67-68-5
T4 RNA Ligase 1 (ssRNA Ligase), High Concentration	New England Biolabs	Cat#M0437M; CAS: 60-00-4
NuPAGE™ LDS Sample Buffer (4×)	Thermo Fisher Scientific	Cat#NP0007; CAS: 56-81-5
ATP, [γ-32P]	PerkinElmer	Cat#BLU002Z250UC
NuPAGE™ 4%–12%, Bis-Tris, 1.0–1.5 mm 10 W, Mini Protein Gels	Thermo Fisher Scientific	Cat#NP0321BOX
Precision Plus Protein Dual Color Standards	Bio-Rad	Cat#1610374
Nitrocellulose Membrane, Roll, 0.45 μm, 30 cm × 3.5 m	Bio-Rad	Cat#1620115; CAS: 9004-70-0
Mini Trans-Blot® Filter Paper, Pkg of 50	Bio-Rad	Cat#1703932
HyBlot CL® Autoradiography Film	Thomas Scientific	Cat#1141J51
Proteinase K Solution (20 mg/mL), RNA grade	Thermo Fisher Scientific	Cat#25530049; CAS: 39450-01-6
Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)	Thermo Fisher Scientific	Cat#AM9722; CAS: 108-95-2, 67-66-3, 123-51-3
Chloroform	J.T.Baker	Cat#9180-01; CAS: 67-66-3
Dynabeads™ MyOne™ Silane	Thermo Fisher Scientific	Cat#37002D
Buffer RLT	QIAGEN	Cat#79216; CAS: 593-84-0
ATP (10 mM)	New England Biolabs	Cat#P0756S; CAS: 1310-73-2
Deoxynucleotide (dNTP) Solution Mix	New England Biolabs	Cat#N0447S
ExoSAP-IT™ PCR Product Cleanup Reagent	Thermo Fisher Scientific	Cat#78200.200.UL
Sodium hydroxide solution (1.0 N, BioReagent, suitable for cell culture)	Sigma-Aldrich	Cat#S2770-100ML; CAS: 1310-73-2
Hydrochloric acid solution (1.0 N, BioReagent, suitable for cell culture)	Sigma-Aldrich	Cat#H9892-100ML; CAS: 7647-01-0
iQ™ SYBR® Green Supermix	Bio-Rad	Cat#1708880; CAS: 56-81-5, 67-68-5
Q5® High-Fidelity 2× Master Mix	New England Biolabs	Cat#M0492S
AMPure XP Reagent	Beckman Coulter	Cat#A63880
Novex™ TBE-Urea Gels, 6%, 12 well	Thermo Fisher Scientific	Cat#EC68652BOX
GelStar™ Nucleic Acid Gel Stain, 10,000×	Lonza	Cat#50535; CAS: 67-68-5
Critical commercial assays
RNA Clean & Concentrator-5 kit	Zymo Research	Cat#R1016
Deposited data
Sequencing data	Biancon et al. (2022),Griffin et al. (2022)	GEO: GSE195620; GSE181264
freCLIP-seq and eCLIP-seq analysis code	Biancon et al. (2022) This paper	GitHub: https://github.com/TebaldiLab/eCLIP_seq https://doi.org/10.5281/zenodo.7121228
Original Images	This paper	Mendeley Data: https://doi.org/10.17632/m7g92hkwmh.1
Experimental models: Cell lines
Human: HEL	ATCC	TIB-180
Experimental models: Organisms/strains
Mouse: Ago2 conditional knockout males (Ddx4-Cre/+;Ago2flox/Δ)	Griffin et al. (2022)	N/A
Oligonucleotides
RNA oligos (X1A/B, X2A/B, RiL19) and DNA oligos (AR17, rand3Tr3)	Van Nostrand et al. (2016)	eCLIP Standard Operating Procedure v1.P 20151108
Illumina Adapters (D501-508, D701-D712)	Illumina Adapter Sequences	https://support-docs.illumina.com/SHARE/AdapterSeq/illumina-adapter-sequences.pdf Pages 50–51. Remember to order the reverse complement of the sequence reported for D701–D712.
Recombinant DNA
CS-TRE-U2AF1-PRE-Ubc-tTA-I2G plasmids	Yamaguchi et al., 2012, Yoshida et al., 2011	N/A
psPAX2	Addgene	Cat#12260
VSV.G	Addgene	Cat#14888
Software and algorithms
FastQC	N/A	https://www.bioinformatics.babraham.ac.uk/projects/fastqc/
FastUniq	Xu et al. (2012)	https://sourceforge.net/projects/fastuniq/
Cutadapt	Martin (2011)	https://cutadapt.readthedocs.io/en/stable/
STAR v2.7.0f	Dobin et al. (2013)	https://github.com/alexdobin/STAR/releases
PureCLIP	Krakau et al. (2017)	https://github.com/skrakau/PureCLIP
Bedtools	Quinlan and Hall (2010)	https://github.com/arq5x/bedtools2
edgeR	Robinson et al. (2010)	https://bioconductor.org/packages/release/bioc/html/edgeR.html
Tidyverse	CRAN	https://www.tidyverse.org/
data.table	CRAN	https://cran.r-project.org/web/packages/data.table/index.html
Ggseqlogo	CRAN	https://cran.r-project.org/web/packages/ggseqlogo/index.html
GenomicAlignments	Bioconductor	https://bioconductor.org/packages/release/bioc/html/GenomicAlignments.html
GenomicRanges	Bioconductor	https://bioconductor.org/packages/release/bioc/html/GenomicRanges.html
Bamsignals	Bioconductor	https://bioconductor.org/packages/release/bioc/html/bamsignals.html
BSgenome	Bioconductor	https://bioconductor.org/packages/release/bioc/html/BSgenome.html
Other
RNAse-free filter tips	USA Scientific	Cat#1121-3810, 1120-1810, 1120-8810, 1126-7810
Low-binding microcentrifuge tubes	Sorenson BioScience	1.7 mL tubes, Cat#11700
Phase Lock Gel Heavy Tubes	Quantabio	2 mL tubes, Cat#2302830
UV crosslinker	Stratagene	UV Stratalinker 2400
Tube rotator	Any brand/model available in the lab	N/A
Sonicator	Branson	Digital Sonifier 450
Thermomixer	Benchmark Scientific	H5000-HC
Magnetic separation rack	Any brand/model available in the lab	N/A
Laboratory cold room	Yale University	Note: For IP and washing steps.
Radioactivity room (only if using γ-32P-ATP, not mandatory)	Yale University	Note: Specific training courses are required. Refer to the safety guidelines of your institution.