Q4. How many R-loop mapping datasets were processed in R-loopBase and how were they processed?

In current release of R-loopBase, we integrated 358 datasets generated in human cells by 16 different technologies (Table 1), 71 datasets in mouse cells by 7 different technologies, 46 datasets in fruit fly cells by 4 different technologies, 23 datasets in yeast cells by 4 different technologies (for meta information please refer to Download). We next developed a comprehensive work-flow for quality control and data analysis. Briefly, technical replicates if existed were merged first, and raw sequencing data were then mapped to the genome (hg38/mm39/dm6/sacCer3) using Bowtie2 local alignment mode. Uniquely-mapped non-redundant reads were kept as useful reads and samples with >7M useful reads were considered as with sufficient read counts. To maximally leverage the sequencing data, biological replicates with <7M useful reads were merged to meet with the minimal reads count cutoff as long as they were highly correlated (Spearman correlation coefficient >0.5). For mouse, fruit fly, and yeast, due to the relatively small data volumes, samples with <7M useful reads were not filtered out. Finally, peak calling was done with MACS2 for all useful reads (DRIP-seq, DRIVE-seq, MapR, R-loop CUT&Tag, R-ChIP-NS, enDR3-ChIP and S1-DRIP-seq) or useful reads from Watson or Crick strand separately (DRIPc-seq, RDIP-seq, ssDRIP-seq, qDRIP-seq, R-ChIP-SS, RR-ChIP, enDR3-DRIPc-seq and BisMapR), using q-value cutoff 0.01 for narrow peak (R-ChIP-SS, R-ChIP-NS, R-loop CUT&Tag and RIAN-seq) and 0.05 for broad peak (DRIP-seq, DRIPc-seq, RDIP-seq, ssDRIP-seq, qDRIP-seq, DRIVE-seq, MapR, RR-ChIP, enDR3-ChIP, enDR3-DRIPc-seq, BisMapR and S1-DRIP-seq). If multiple biological replicates existed, peaks with ≥50bp overlap among ≥2 replicates were merged and taken as reproducible peaks. Human samples with <100 peaks, mouse samples with <50 peaks, fruit fly and yeast samples with <10 peaks called were discarded. Following ENCODE guidelines for ChIP-seq data analyses, we further calculated signal portion of tags (SPOT) and reads in blacklisted regions (RiBL) as part of quality control matrix for users' reference. Only peaks outside of ChIP-seq blacklisted regions were used for downstream analysis. Specially, bisDRIP-seq and spKAS-seq data are not readily for peak calling, we instead uploaded their processed signal tracks onto our genome browser for visualization and comparison with other R-loop mapping data. In total, 358 datasets for 55 human cells generated by 16 different technologies (Table 1), 71 datasets for 17 mouse cells generated by 7 different technologies (Table 2), 46 datasets for 16 fruit fly cells generated by 4 different technologies (Table 3), 23 datasets for 5 yeast cells generated by 4 different technologies (Table 4), have been included in current release of R-loopBase .

Table 1. Meta information for Human R-loop mapping data

Technology	Treatment	Biological Samples	Datasets	PMID
DRIP-seq	Control	B-cell (1/1*), CHLA10 (1/1), EWS502(1/1), HeLa (10/9), HEK293 (2/2), SHSY5Y (2/2), TC32 (1/1), Stromal (4/4), Basal-epithelial (4/4), Luminal-progenitor (4/4), Mature-luminal-epithelial (4/4), MCF-7 (1/1), NT2 (6/6), K562 (3/1), Primary-fibroblast (2/2), U2OS (18/14), U87 (2/2), Jurkat (2/0), T-cells (2/0), IMR-90 (1/0), HEK293T (3/2), A375 (1/1), M231 (4/4), CD34+ (4/2), CD4+-HD (1/1), DMS114 (1/1), HCT116 (4/4), MCF10A (2/1), MDA-MB-436 (2/2), MOLM13 (3/2), Nalm-6 (2/2), Neuroblastoma (6/6), PC-9 (2/2), SNB-19 (3/2), Sperm (4/1), U251 (1/1), Blood (2/2), foreskin-fibroblast (1/0), KM27 (1/0), HFK (2/0), CIN612 (2/0), tIMEC (4/0)	126/95	30108179, 32669707, 32769985, 28802045, 30060749, 28270613, 28649985, 27552054, 27373332, 23868195, 22387027, 26182405, 32747416, 30591567, 29416069, 29416038, 32439635, 32398827, 32686621, 28341774, 32615088, 38267456, 38200551, 38787953, 36453989, 38184854, 36999631, 35618715, 33661429, 38589367, 39178326, 34624217, 37831098, 35180428, 33986538, 37697435, 37139234, 37735199, 37463047, 37777505, 35929179, 37010886, 38761375, 40138394, 39828096, 40108134, 41129225, 40836041, 39779698, 40386679, 40382323
	Knock down	U2OS (17/12) , U87 (2/2), HeLa (9/8), HEK293 (2/2), SHSY5Y (2/2), M231 (6/0), MOLM13 (2/0), KM27 (1/0), tIMEC (4/0), MCF10A (1/1), U251 (1/1), HEK293T (2/2), DMS114 (1/1), MDA-MB-436 (4/4), A375 (1/1), Nalm-6 (2/2), PC-9 (2/2), CD34+ (2/1), Neuroblastoma (6/6)	67/47	32747416, 32669707, 32686621, 32769985, 30060749, 28270613, 38200551, 38184854, 36999631, 35618715, 33661429, 38589367, 34624217, 37831098, 35180428, 37697435, 37735199, 37463047, 35929179, 38761375, 40138394, 39828096, 40108134, 41129225, 39779698, 40386679, 40382323
RDIP-seq	Control	HeLa (2/2), IMR-90 (1/1), HEK293T (1/0)	4/3	30449723, 26579211
	Knock down	HeLa (2/2)	2/2	30449723
DRIPc-seq	Control	K562 (3/3), HEK293 (2/2), NT2 (2/2), CD4+-AD (3/3), MOLM13 (2/1)	12/11	32439635, 30060749, 27373332, 40823807, 35180428, 33986538, 40836041
	Knock down	K562 (4/4), HEK293 (2/2), MOLM13 (2/0)	8/6	32439635, 30060749, 35180428, 33986538
ssDRIP-seq	Control	HeLa (3/3), hVECs (2/2), hESCs (2/2), hiPSCs (2/2), hMSCs (2/2), hNSCs (2/2), hVSMCs (2/2)	15/15	31606733, 32640435
	Knock down	HeLa (3/3)	3/3	31606733
qDRIP-seq	Control	HeLa (3/2), U2OS (2/2)	5/4	32544226, 40447771
	Knock down	U2OS (1/1)	1/1	40447771
R-ChIP-SS	Control	HEK293T (5/5), K562 (2/2), HeLa (1/0), HEK293 (2/2), HUH7 (1/0)	11/9	29104020, 32966794, 39019869, 40790042
	Knock down	HEK293 (2/2)	2/2	39019869
MapR	Control	HEK293 (3/3), U87T (2/2), HeLa (2/2), SKM-1 (1/1), CUTLL1 (6/6), hiPSC (2/2), HCT116 (3/3), U2OS (3/3)	22/22	31665646, 34916496, 35061527, 35013239, 40037355, 40613709
	Knock down	HCT116 (3/3), U2OS (3/3), hiPSC (2/2), SKM-1 (1/1), HeLa (2/2), CUTLL1 (2/2)	13/13	34916496, 35061527, 35013239, 40037355, 40613709
R-loop CUT&Tag	Control	HEK293T (7/6), HEK293 (1/1), OMM2.3 (1/1), DLD-1 (2/0), HeLa (4/0)	15/8	33597247, 37557913, 34232287, 37270643, 38858601, 40579572
	Knock down	DLD-1 (2/2), HEK293T (2/0), OMM2.3 (1/0)	5/2	37557913, 34232287, 37270643
RIAN-seq	Control	HEK293T (4/4), HeLa (2/2)	6/6	40112807
	Knock down	HEK293T (2/2)	2/2	40112807
R-ChIP-NS	Control	U2OS (2/2)	2/2	38717338
	Knock down	U2OS (2/0)	2/0	38717338
bisDRIP-seq	Control	MCF-7 (13/13)	13/13	29072160
DRIVE-seq	Control	NT2 (1/1)	1/1	22387027
RR-ChIP	Control	HeLa (2/2)	2/2	31679819
spKAS-seq	Control	HEK293 (6/6), HEK293T (2/2), HepG2 (2/2), K562 (2/2)	12/12	39351875, 36449625
enDR3-ChIP	Control	HeLa (1/1)	1/1	40823807
enDR3-DRIPc-seq	Control	HeLa (3/3)	3/3	40823807
SUM	-	-	358/285	-

^*number of datasets analyzed / high-quality datasets.
Table 2. Meta information for Mouse R-loop mapping data

Technology	Treatment	Biological Samples	Datasets	PMID
DRIP-seq	Control	3T3 (1/1), E14 (1/1), HEPA1-6 (2/2), Liver (2/2), N2a (3/3), MEF (1/1)	10/10	38219817, 27373332, 33357438, 39369271
	Knock down	MEF (1/1) , N2a (6/6)	7/7	38219817, 33357438
CUT&Tag	Control	CD4+-naive-T-cell (2/1), Leptotene-zygotene (3/3), mESC (4/4), Pachytene-diplotene (3/2), Spermatogonia (3/3), Spleen (2/2), Secondary spermatocytes (3/0)	20/15	36396044, 40504899, 40911696, 41417894
	Knock down	mESC (8/8), Spleen (2), CD4+-naive-T-cell (2/0)	12/10	40504899, 40911696, 41417894
ssDRIP-seq	Control	Retina (4/2), Testis (2/2), iPS (2/2), MEF (2/2)	10/8	32704541, 39824811, 40047526
	Knock down	Testis (2/2)	2/2	39824811
RIAN-seq	Control	mESC (2/2)	2/2	40112807
DRIPc-seq	Control	3T3 (1/1)	1/1	27373332
MapR	Control	mESC (2/2), B-cells (3/3)	5/5	34937926, 33620319
BisMapR	Control	mESC (2/2)	2/2	33620319
SUM	-	-	71/62	-

Table 3. Meta information for Fruit Fly R-loop mapping data

Technology	Treatment	Biological Samples	Datasets	PMID
CUT&Tag	Control	Embryo-E10-14h (1/1), Embryo-E14-16h (4/4), Embryo-E20-22h (2/2), Embryo-E2-4h (2/2), Embryo-E8-10h (2/2), Larva-L3 (2/2)	13/13	39470713
	Knock down	Embryo-E14-16h (2/2)	2/2	39470713
ssDRIP-seq	Control	Brain (2/2)	2/2	40660165
	Knock down	Brain (2/2)	2/2	40660165
DRIP-seq	Control	10-14H-Oregon-R-embryos (2/2), 2-6H-Oregon-R-embryos (2/2), DGRP-379-female (2/2), DGRP-379-male (2/2), DGRP-732-female (2/2), DGRP-732-male (2/2), S2 (4/4), w1118-ovaries (2/2)	18/18	32286294, 28819201, 40036798
MapR	Control	Photoreceptor-neuron (9/9)	9/9	35048512
SUM	-	-	46/46	-

Table 4. Meta information for Yeast R-loop mapping data

Technology	Treatment	Biological Samples	Datasets	PMID
DRIP-seq	Control	S288C (2/2), WRBb-9D (1/1), BY4741 (1/0)	4/3	29954833, 25357144, 27638543, 40064914
	Knock down	BY4741 (2/2)	2/2	25357144
S1-DRIP-seq	Control	S288C (4/4)	4/4	27298336
	Knock down	S288C (4/4)	4/4	27298336
DRIPc-seq	Control	W303-G1 (1/1), W303-S (2/2)	3/3	34294712
	Knock down	W303-S (4/4)	4/4	34294712
RIAN-seq	Control	S288C (2/2)	2/2	40112807
SUM	-	-	23/22	-