Single-cell sequencing of individual retinal organoids reveals determinants of cell-fate heterogeneity

. 2023 Aug 9;3(8):100548. doi: 10.1016/j.crmeth.2023.100548

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

POU4F2	Santa Cruz Biotechnology	Cat# SC-6026; RRID: AB_673441
PAX2	BioLegend	Cat# 901001; RRID: AB_2565001
VSX2	Exalpha Biologicals	Cat# X1180P; RRID: AB_2314191
VSX2	SantaCruz	Cat# Sc-365519; RRID:AB_10842442
MAP2	Sigma-Aldrich	Cat# M9942; RRID:AB_477256
OTX2	Abcam	Cat# ab21990; RRID:AB_776930
ELAVL3/4	Thermo Fisher Scientific	Cat# A-21271; RRID:AB_221448
RCVRN	MIllipore	Cat# AB5585; RRID:AB_2253622
Donkey anti-goat 488 (1:250 dilution)	Thermo Fisher Scientific	Cat# A-11055; RRID:AB_2534102
Donkey anti-rabbit 647 (1:250 dilution)	Thermo Fisher Scientific	Cat# A-31573; RRID:AB_2536183
Donkey anti-sheep 647 (1:300 dilution)	Thermo Fisher Scientific	Cat# A-21448; RRID:AB_2535865
Donkey anti-mouse 488 (1:250 dilution)	Jackson ImmunoResearch Labs	Cat# 715-546-151; RRID:AB_2340850
Donkey anti-mouse 568 (1:250 dilution)	Thermo Fisher Scientific	Cat# A10037; RRID:AB_2534013
Donkey anti-goat 568 (1:250 dilution)	Thermo Fisher Scientific	Cat# A-11057; RRID:AB_2534104
Donkey anti-rabbit 647 (1:250 dilution)	Thermo Fisher Scientific	Cat# A-31573; RRID:AB_2536183

Chemicals, peptides, and recombinant proteins

Matrigel	Corning	CLS354277
ReLeSR	StemCell Technologies	100–0484
BMP4	R&D Systems	314-BP-050
CHIR99021	Cedarlane Laboratories	04-0004-02
SU5402	Cayman Chemical Co	13182
SAG	Calbiochem	566660
DPBS (no calcium, no magnesium)	ThermoFisher	14190144
Accutase	Sigma	A6964-100ML
FBS	Corning	35-011-CV
Papain and Ovomucoid	Worthington Biochemical	LK003150
Fluromount-G	SouthernBiotech	0100–01
IGEPAL	Sigma-Aldrich	I8896
SuperaseIn RNase Inhibitor	Ambion	AM2694
BSA	NEB	B9000S
5% paraformaldehyde	EMS	50-980-493
Triton X-100	ThermoFisher	A16046.AP
4′,6-diamidino-2-phenylindole (FACS)	Invitrogen	D1306
4′,6-diamidino-2-phenylindole (Immunostaining)	Sigma	D9542
EB buffer	Qiagen	19086
mRNA Second Strand Synthesis buffer	NEB	E6111L
N7 loaded custom TDE1 enzyme	MacroLab, UC Berkeley	N/A
Dimethyl Formamide	Sigma-Aldrich	227056-100ML
DNA binding buffer	Zymo Research	D4004-1-L
D1000 Screen Tape	Agilent Technologies	5067–5583
DNA purification	Zymo Research	D4014
AMPure XP Reagent	Beckman Coulter Life Sciences	A63881

Critical commercial assays

Single Cell 3′ v3.1 Dual Index Gene Expression kit (Dual Index Kit TT Set A 96 rxns)	10x Genomics	1000215

Deposited data

Raw and analyzed data	This paper	GEO: GSE220661

Experimental models: Cell lines

Human: H9 hESCs NRL+/eGFP	Phillips³⁸	N/A

Oligonucleotides

Hashing oligo: 5′-GTCTCGTGGGCTCGGAGATG TGTATAAGAGACAG-[10bp-barcode]-BAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAA-3′ where B is G, C or T	Srivatsan et al.²⁶	N/A
Indexed 2-level RT oligo: oligo-dT primer 5ʹ-ACGA CGCTCTTCCGATCTNNNNNNNN-[10bp index]-TT TTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3ʹ, where N is any base and V is either A, C or G	Cao et al.⁴⁵	N/A
Indexed 3-level RT oligo: (5’-[PHO]- CAGAGCNNN NNNNN-[10-bp barcode]-TTTTTTTTTTTTTTTTTTT TTTTTTTTTTT-3′ where N is any base	Cao et al.³⁴	N/A
Indexed 3-level ligation oligo: 5′-GCTCTG-[9-bp or 10-bp barcode A]-/dideoxyU/ACGACGCTCTT CCGATCT-[reverse complement of barcode A]-3′	Cao et al.³⁴	N/A
Indexed P5 PCR oligo: 5′-AATGATACGGCGACC ACCGAGATCTACAC-[i5]-ACACTCTTTCCCTACA CGACGCTCTTCCGATCT-3′	Cao et al.⁴⁵	N/A
Indexed P7 PCR oligo: 5′-CAAGCAGAAGACGGC ATACGAGAT-[i7]-GTCTCGTGGGCTCGG-3′	Cao et al.⁴⁵	N/A

Software and algorithms

ImageJ	Schneider et al.⁴⁶	https://imagej.nih.gov/ij/
10x Genomics Cell Ranger	Zheng et al.⁴⁷	https://support.10xgenomics.com/single-cell-gene-expression/software/downloads/latest
Monocle3	Cao et al.³⁴	https://cole-trapnell-lab.github.io/monocle3/
BBI pipeline is available at the bbi-lab github page under the bbi-dmux and bbi-sci repositories	NA	https://github.com/bbi-lab
Seurat	Stuart et al.⁴⁸	https://satijalab.org/seurat/
VGAM	Yee⁴⁹	https://CRAN.R-project.org/package=VGAM
Code related to this paper	This paper	https://zenodo.org/badge/latestdoi/654269063

Other

Keyence BZ-X810 microscope	Keyence	N/A
Zeiss Axio Observer D1	Zeiss	N/A
Zeiss LSM 990	Zeiss	N/A
1.5 mL LoBind microcentrifuge tube	Eppendorf	Z666491
twin.tec™ 96 Well LoBind PCR Plates	Eppendorf	0030129512
Bioruptor Plus	diagenode	B01020001