Reagent/Resource	Reference or Source	Identifier or Catalog Number
Experimental Models
D. melanogaster Schneider S2 cells	Thermo Fisher	Cat#R69007
HCT116	ATCC	Cat#CCL‐247
Parental OsTir expressing S2 cell line	This study	N/A
TRF2 C‐terminally tagged AID S2 cell line	This study	N/A
TBP N‐terminally tagged AID S2 cell line	This study	N/A
DREF N‐terminally tagged AID S2 cell line	This study	N/A
TFIIA C‐terminally tagged AID S2 cell line	This study	N/A
Chromator N‐terminally tagged AID S2 cell line	This study	N/A
Recombinant DNA
pBabe Puro osTIR1‐9Myc	Addgene	plasmid #80074
pAc‐sgRNA‐Cas9	Addgene	plasmid #49330
pCRIS‐PITChv2‐FBL	Addgene	plasmid #63672
pGL13_tGFP	This study	N/A
Antibodies
Mouse monoclonal anti‐FLAG	Sigma‐Aldrich	Cat#F3165
Secondary anti‐mouse HRP	Sigma‐Aldrich	Cat#12‐349
Histone H3	Abcam	Cat#ab1791
Alpha‐tubulin	Abcam	Cat#Ab18251
Secondary anti‐rabbit HRP	Sigma‐Aldrich	Cat#12‐348
Oligonucleotides and other sequence‐based reagents For long lists of oligos or other sequences please refer to the relevant Table(s) or EV Table(s)
5′‐ /5Phos/rNrNrN rNrNrN rNrNrG rArUrC rGrUrC rGrGrA rCrUrG rUrArG rArArC rUrCrU rGrArA rC/3InvdT/‐3′ (3′ RNA linker)	IDT	N/A
5‐rCrCrU rUrGrG rCrArC rCrCrG rArGrA rArUrU rCrCrA rNrNrN rN ‐3 (5′ RNA linker)	IDT	N/A
Biotin TEG 5′ [BtnTg]GCAGGTGCCAGAACATTTCTCTATCGATAGG	Sigma‐Aldrich	N/A
Reverse 3′ CTTTACCAACAGTACCGGAATGC	Sigma‐Aldrich	N/A
Act5C gRNA forward TTCGGACCGCAAGTGCTTCTAAGA	Sigma‐Aldrich	N/A
Act5C gRNA reverse AACTCTTAGAAGCACTTGCGGTC	Sigma‐Aldrich	N/A
TBP N‐terminus gRNA forward TTCGACAATAAACCATCTGTAAGA	Sigma‐Aldrich	N/A
TBP N‐terminus gRNA reverse AACTCTTACAGATGGTTTATTGTC	Sigma‐Aldrich	N/A
DREF N‐terminus gRNA forward ttcGGAAGACAAGATGAGCGAAG	Sigma‐Aldrich	N/A
DREF N‐terminus gRNA reverse aacCTTCGCTCATCTTGTCTTCC	Sigma‐Aldrich	N/A
Chromator N‐terminus gRNA forward TTCGCTGGAGTCGTGAATAATGT	Sigma‐Aldrich	N/A
Chromator N‐terminus gRNA reverse AACACATTATTCACGACTCCAGC	Sigma‐Aldrich	N/A
TFIIA‐L C‐terminus gRNA forward TTCGCGACGCCGAGTGGTAATGGA	Sigma‐Aldrich	N/A
TFIIA‐L C‐terminus gRNA reverse AACTCCATTACCACTCGGCGTCGC	Sigma‐Aldrich	N/A
TBP AID N‐terminal repair cassette forward CCGCGTTACATAGCATCGTACGCGTACGTGTTTGGTCCACAATAAACCATCTGTAATGGCCAAGCCTTTGTCTCAAG	Sigma‐Aldrich	N/A
TBP AID N‐terminal repair cassette reverse CATCAGCATTCTAGAGCATCGTACGCGTACGTGTTTGGCTTAGCATTTGGTCCATCTGCGAGCCACCGCCCGATC	Sigma‐Aldrich	N/A
DREF AID N‐terminal repair cassette forward ccgcgttacatagcatcgtacgcgtacgtgtttggCACAGAAGACAAGATGAGCGATGGCCAAGCCTTTGTCTCAAG	Sigma‐Aldrich	N/A
DREF AID N‐terminal repair cassette reverse catcagcattctagagcatcgtacgcgtacgtgtttggGGGCGACGCTGGTACCCCTTCCGAGCCACCGCCCGATC	Sigma‐Aldrich	N/A
TFIIA‐L AID C‐terminal repair cassette forward CCGCGTTACATAGCATCGTACGCGTACGTGTTTGGCGAATGGCGACGCCGAGTGGGGCGGTGGCTCGGGAG	Sigma‐Aldrich	N/A
TFIIA‐L AID C‐terminal repair cassette reverse CATCAGCATTCTAGAGCATCGTACGCGTACGTGTTTGGTGTTCGCTCAACTGCCATCCTTAGCCCTCCCACACATAACCAG	Sigma‐Aldrich	N/A
Chromator AID N‐terminal repair cassette forward gttccgcgttacatagcatcgtacgcgtacgtgtttggGGCGCTGGAGTCGTGAATAAATGGCCAAGCCTTTGTCTCA	Sigma‐Aldrich	N/A
Chromator AID N‐terminal repair cassette reverse catcagcattctagagcatcgtacgcgtacgtgtttggTGAAATCTCCTGTGCCAACATCGAGCCACCGCCCGATC	Sigma‐Aldrich	N/A
OsTir ligase donor cassette forward TGGATCTCCAAGCAGGAGTACGACGAGTCCGGCCCCTCCATTGTGCACCGCAAGTGCTTCGGCAGCGGCGCCAC	Sigma‐Aldrich	N/A
OsTir ligase donor cassette reverse CCTCCAGCAGAATCAAGACCATCCCGATCCTGATCCTCTTGCCCAGACAAGCGATCCTTCCTAGCCCTCCCACACATAACCAG	Sigma‐Aldrich	N/A
Genotyping Act5C OsTir forward GGCTTCGCTGTCCACCTTCCAG	Sigma‐Aldrich	N/A
Genotyping Act5C OsTir reverse GAAGTCGAGGAAGCAGCAGCGA	Sigma‐Aldrich	N/A
Chemicals, enzymes, and other reagents (e.g., drugs, peptides, recombinant proteins, and dyes)
4–20% Mini‐PROTEAN® TGX™ Precast Protein Gels, 15‐well, 15 μl	Bio‐Rad	Cat#34561096
MegaX DH10B T1R Electrocomp™ Cells	Thermo Fisher	Cat#C640003
FastDigest MluI	Thermo Fisher	Cat#FD0564
BspQI	NEB	Cat#R0712S
Blasticidin S HCl	Thermo Fisher	Cat#R21001
3‐Indoleacetic acid	Merck	Cat#I3750
QuickExtract™ DNA Extraction Solution	Lucigen	Cat#QE9059
2× Laemmli Sample Buffer	Bio‐Rad	Cat#1610737
EGTA	Merck	Cat#E4378
Biotin‐11‐CTP	PerkinElmer	Cat#NEL542001EA
Biotin‐11‐UTP	PerkinElmer	Cat#NEL543001EA
Q5 polymerase high‐fidelity 2× master mix	NEB	Cat#M0492S
Trizol	Thermo Fisher	Cat#15596026
Trizol‐LS	Thermo Fisher	Cat#10296010
GlycoBlue™ Coprecipitant	Thermo Fisher	Cat#AM9515
NTP Set, 100 mM Solution	Thermo Fisher	Cat#R0481
N‐Lauroylsarcosine sodium salt	Merck	Cat#L5125
Dynabeads™ M‐280 Streptavidin	Thermo Fisher	Cat#11205D
Cap‐CLIP	BioZym	Cat#C‐CC15011H
T4 Polynucleotide Kinase	NEB	Cat#M0201S
Murine RNAse Inhibitor	NEB	Cat#M0314L
T4 RNA Ligase	NEB	Cat#M0204L
SuperScript™ III Reverse Transcriptase	Thermo Fisher	Cat#18080093
KAPA HiFi HotStart Real‐Time Library Amp Kit	Roche	Cat#7959028001
AMPure XP beads	Beckman Coulter	Cat#A63882
Anti‐FLAG® M2 Magnetic Beads	Merck	Cat#M8823
Lysyl endopeptidase	Wako Chemicals	Cat#7041
Ammoniumbicarbonate	Sigma‐Aldrich	Cat#09830
Tris‐(2‐carboxyethyl)‐phosphin‐hydrochloride (TCEP)	Sigma‐Aldrich	Cat#646547
S‐Methyl‐thiomethanesulfonate (MMTS)	Sigma‐Aldrich	Cat#64306
Trifluoroacetic acid	Sigma‐Aldrich	Cat#T6508
oComplete mini protease inhibitors	Sigma‐Aldrich	Cat# 11836170001
Axygen 1.5 ml MaxyClear tube	Corning	Cat#MCT‐150‐A
Axygen 0.6 ml MaxyClear tube	Corning	Cat#MCT‐060‐C‐S
Direct‐zol RNA Microprep	Zymo	Cat#R2061
Micro Bio‐spin P‐30 gel columns	Bio‐rad	7326251
Software Include version where applicable
MSAmanda	N/A	https://ms.imp.ac.at/?goto=msamanda
Benchling	N/A	https://benchling.com
R version 3.5.3	R Development Core Team, 2019	https://www.r‐project.org
Cutadapt	Martin (2011)	https://bioweb.pasteur.fr/packages/pack@cutadapt@1.18
Samtools version 1.9	Li et al, 2009	http://www.htslib.org/
bowtie version 1.2.2	Langmead et al, 2009	https://sourceforge.net/projects/bowtie‐bio/files/bowtie/1.2.2/
GenomicRanges 1.34.0	Lawrence et al, 2013	https://bioconductor.org/packages/release/bioc/html/GenomicRanges.html
Biostrings 2.50.2	N/A	https://bioconductor.org/packages/Biostrings
bigBedtoBed	Kent et al, 2010	https://github.com/ENCODE‐DCC/kentUtils/blob/master/src/utils/bigBedToBed/bigBedToBed.c
bedtools 2.27.1	Quinlan & Hall, 2010	https://github.com/arq5x/bedtools2/releases/tag/v2.30.0
DESeq2 package v.1.30.1	Love et al, 2014	https://bioconductor.org/packages/release/bioc/html/DESeq2.html
Other
Mini‐PROTEAN Tetra Vertical Electrophoresis Cell	Bio‐Rad	Cat#1658004
Monarch Gel Extraction	NEB	Cat#T1020L
Illumina Truseq small RNA library prep kit	Illumina	Cat#RS‐200‐0012
Power Blotter Station	Thermo Fisher	Cat#PB0010
MaxCyte STX Scalable Transfection System	Maxcyte	NA