REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit polyclonal anti-Phospho-Histone H2A.X (Ser139)	Abcam	Cat#ab2893; RRID: AB_303388
Rabbit monoclonal anti-Phospho-Histone H3 (Ser10)	Cell Signaling Technology	Cat#3377S; RRID: AB_1549592
Rabbit polyclonal anti-Histone H3	Abcam	Cat#ab1791; RRID: AB_302613
Rabbit polyclonal anti-Histone H2A.X	Merck-Millipore	Cat#07-627
Rabbit polyclonal anti-Histone H3	Cell Signaling Technology	Cat#9715S; RRID: AB_331563
Mouse monoclonal anti-BrdU	Sigma-Aldrich	Cat#B8434; RRID: AB_476811
Mouse monoclonal anti-Tubulin	The Francis Crick Institute Core Facility	Tat-1
Mouse monoclonal anti-BLM	The Francis Crick Institute Core Facility	BFL103
Mouse monoclonal anti Ser5-RPB1	The Francis Crick Institute Core Facility	4H8
Mouse monoclonal anti-U1 snRNP 70	Santa Cruz Biotechnoloy	Cat#sc-390988
Mouse monoclonal anti-NELF-A	Santa Cruz Biotechnoloy	Cat#sc-365004; RRID: AB_10708864
Mouse monoclonal anti-SPT5	Santa Cruz Biotechnoloy	Cat#sc-133217; RRID: AB_2196394
Rabbit polyclonal anti-RAD51	Millipore	Cat#PC130
Mouse anti-Biotin	Jackson ImmunoResearch	Cat#200-002-211; RRID:AB_2339006
Rabbit anti-Biotin	Bethyl Laboratories	Cat#A150-109A; RRID:AB_67327
HRP-linked Horse anti-mouse IgG	Cell Signaling Technology	Cat#7076S; RRID: AB_330924
HRP-linked Goat anti-rabbit IgG	Cell Signaling Technology	Cat#7074S; RRID: AB_2099233
FITC-conjugated Goat anti-Mouse IgG	Sigma-Aldrich	Cat#F2012; RRID: AB_259456
Alexa Fluor488-conjugated Goat Anti-Rabbit IgG (H+L)	Thermo Fisher Scientific	Cat#A-11070; RRID: AB_2534114
Chemicals, peptides, and recombinant proteins
RNase A	AppliChem	Cat#A2760,0100
SUPERase.In	Thermo Fisher Scientific	Cat#AM2696
BrdU	Sigma-Aldrich	Cat#B5002
EdU	Sigma-Aldrich	Cat#900584
Nocodazole	Sigma-Aldrich	Cat#M1404
Ro 3306	Adooq Bioscience	Cat#A14437
Pepsin	Sigma-Aldrich	Cat#P6887
Propidium iodide	Sigma-Aldrich	Cat#P4170
Violet Stain	Thermo Fisher Scientific	Cat#V35003
DRB	Sigma-Aldrich	Cat#D1916
Triptolide	Sigma-Aldrich	Cat#T3652
Quick Start Bradford protein 1x dye reagent	Bio-Rad	Cat#5000205
Agencourt AMPure XP	Beckman Coulter	Cat#A63881
Dynabeads Protein A	Thermo Fisher Scientific	Cat#10002D
Fluoroshield with DAPI	GeneTex	Cat#GTX30920
INTERFERin siRNA Transfection Reagent	Polyplus	Cat#409-10
ATM inhibitor	Stratech	Cat#KU-55933
ATR inhibitor	Stratech	Cat#AZD6738
Camptothecin	Sigma-Aldrich	Cat#208925
Etoposide	Sigma-Aldrich	Cat#E1383
CD437	Sigma-Aldrich	Cat#C5865
Rad51 inhibitor (B02)	Sigma-Aldrich	Cat#SML0364
Rad52 inhibitor (AICAR)	Sigma-Aldrich	Cat#A9978
PARP inhibitor (Olaparib)	Adooq Bioscience	Cat#10111
Aphidicolin	Sigma-Aldrich	Cat#A4487
Critical commercial assays
PureLink Genomic DNA Mini Kit	Beckman Coulter	Cat#A63881
DNA Clean & Concentrator-5	Thermo Fisher Scientific	Cat#10002D
RNeasy Mini Kit	GeneTex	Cat#GTX30920
RNA Clean & Concentrator-5	Polyplus	Cat#409-10
Ribo-Zero Gold rRNA Removal Kit (H/M/R)		Cat#MRZG12324
Random Primed DNA Labeling Kit	Thermo Fisher Scientific	Cat#K182001
Qubit dsDNA HS Assay Kit	Zymo Research	Cat#D4013
SensiFAST SYBR Lo-ROX Kit	QIAGEN	Cat#74106
Click-iT RNA Alexa Fluor 594 Imaging Kit	Zymo Research	Cat#R1015
Nextera XT DNA Library Preparation Kit	Bioline	Cat#BIO-94020
QIAseq Ultralow Input Library Kit	Thermo Fisher Scientific	Cat#C10330
KAPA RNA HyperPrep Kit with RiboErase (HMR)	Sigma-Aldrich	Cat#DUO92002
KAPA Library Quantification Kit	Sigma-Aldrich	Cat#DUO92004
ECL Western Blotting Substrate	Sigma-Aldrich	Cat#DUO92008
SuperScript III Reverse Transcriptase kit	Illumina	Cat#FC-131-1024
Deposited data
All the datasets generated in this paper	This paper	GSE136294
BJ GRO-Seq	Li et al., 2018	SRX3586216
CTR cells and RNAi of NELFB and NELFE GRO-Seq	Liu et al., 2017	GSE98555
RPE EdU-Seq	Macheret and Halazonetis, 2018	PRJNA397123
U2OS EdU-Seq	Macheret and Halazonetis, 2018	PRJNA397123
CAP-Seq HeLa cells	Tan-Wong et al., 2019	GSE87607
Ok-Seq RPE h-TERT	Chen et al., 2019	GSE114017
Ok-Seq HeLa	Petryk et al., 2016	SRP065949
ChIP-Seq ORC1 HeLa	Dellino et al., 2013	GSE37583
ChIP-Seq MCM7 HeLa	Sugimoto et al., 2018	GSE107248
ChIP-Seq RPA2 HeLa	Zhang et al., 2017	GSE76661
ChOR-Seq mouse ES cells	Stewart-Morgan et al., 2019	GSE128643
HeLa polyA RNA-Seq	Encode Project Consortium	ENCFF343WEZ
ChIP-Seq 53BP1, RAD51, XRCC4 U2OS	Clouaire et al., 2018	E-MTAB-5817
Experimental models: cell lines
Human osteosarcoma (U2OS)	ATCC	HTB-96
Human immortalized fibroblasts (BJ-hTERT)	ATCC	CRL-4001
Oligonucleotides
siGENOME Non-Targeting siRNA Pool #2	Dharmacon	Cat#D-001206-14-05
siGENOME Human SUPT5H siRNA Pool	Dharmacon	Cat#M-016234-01-0005
siGENOME Human NELFA siRNA Pool	Dharmacon	Cat#M-012156-00-0005
Nextera XT Index Kit	Illumina	Cat#FC-131-1001
GeneRead Adaptor I Set A 12-plex (144)	QIAGEN	Cat#180985
SeqCap Adaptor Kit A	Roche	Cat#07141530001
See Table S5 for Real Time-PCR primer list		n/a
Software and algorithms
EaSeq	Lerdrup et al., 2016	https://easeq.net
R	Friendly, 2002	https://www.r-project.org
Bioconductor	Gentleman et al., 2004	https://bioconductor.org
Bowtie 2	Langmead and Salzberg, 2012	http://bowtie-bio.sourceforge.net/bowtie2/index.shtml
SAMtools	Li et al., 2009	http://www.htslib.org
STAR	Dobin et al., 2013	https://github.com/alexdobin/STAR
BBDuk	Bushnell, BBTools Team	https://github.com/BioInfoTools/BBMap/blob/master/sh/bbduk.sh
featureCounts	Liao et al., 2014	https://www.rdocumentation.org/packages/Rsubread/versions/1.22.2/topics/featureCounts
Galaxy	Afgan et al., 2018	https://usegalaxy.org
MACS2	Zhang et al., 2008	https://github.com/macs3-project/MACS
Bedtools	Quinlan and Hall, 2010	https://bedtools.readthedocs.io/en/latest/
deepTools	Ramírez et al., 2014	https://deeptools.readthedocs.io/en/develop/
pybedtools	Dale et al., 2011	https://daler.github.io/pybedtools/
Prism	GraphPad	Version 7
IGV	Robinson et al., 2011	https://software.broadinstitute.org/software/igv/
FlowJo	Becton Dickinson	Version 10.6.1