Stabilization of hESCs in two distinct substates along the continuum of pluripotency

. 2022 Nov 2;25(12):105469. doi: 10.1016/j.isci.2022.105469

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

mouse anti-OCT4	Santa-Cruz	sc-5279
mouse anti-SSEA4	cell signaling technology	CST-4755S
mouse anti-TRA-1-60	Abcam	ab16288
rabbit anti-TFE3	Sigma-Aldrich	HPA023881

Biological samples

Parental DNA/RNA for Lis38/39 and Lis45/4	This paper	N/A

Chemicals, peptides, and recombinant proteins

Neurobasal medium	Gibco	21103-049
DMEM/F-12	Gibco	21331-020
Pen/Strep	Biological industries	03-033-1B
L-Glutamine	Biological industries	03-020-1B
1% non-essential amino acids	Biological industries	01-340-1B
sodium pyruvate	Biological industries	03-042-1B
B27 supplement	This paper	N/A
0.2% ml defined lipid concentrate	Gibco	11905-031
β-ME	Gibco	31350-010
Insulin	Sigma-Aldrich	I-1882
apo-transferrin	Sigma-Aldrich	T-1147
progesterone	Sigma-Aldrich	P8783
Putrescine	Sigma-Aldrich	P5780
sodium selenite	Sigma-Aldrich	S5261
L-ascorbic acid 2-phosphate	Sigma-Aldrich	A8960
BSA	Gibco	1526-037
Human LIF	Peprotech	300-05
IWR1	Biotest	3532
human activin A	Peprotech	120-14E
FGF2	Peprotech	100-18B
Chir99021	Axon Medchem	1386
PD0325901	Axon Medchem	1408
BIRB796	Axon Medchem	1358
SP600125	Tocris	1496
PKCi	Tocris	2285
Thiazovivin	Peprotech	1535
CGP77675	Axon Medchem	2097
20% Knockout Serum Replacement	Gibco	10828028
trypsin + EDTA	Biological industries	03-053-1B
Y27632 ROCK inhibitor	Axon Medchem	1683
Triton X-100	Sigma-Aldrich	X100
FxCycle Violet	Invitrogen	F10347
Colcemid	Biological industries	12-004-1D
Ampure XP beads	Beckman Coulter	A63881

Critical commercial assays

RNeasy mini kit	Qiagen	74104
Superscript IV RT-PCR kit	Invitrogen	18091050
FAST SYBR Green Master Mix	Quanta bio	95072-012
Click-iT EdU Alexa Fluor 647 Flow Cytometry Kit	Life Technologies	C10420
AP staining kit	Stemgent	00-0055
DNeasy Blood & Tissue Kit	Qiagen	20-69504
Kapa HyperPlus Kit	Roche	KK8510
Qubit dsDNA BR	ThermoFisher Scientific	Q32853
Agilent DNA 7500 Kit	Agilent	5067-1506
mirVana miRNA Isolation Kit	ThermoFisher Scientific	AM1560
KAPA mRNA HyperPrep Kit	Roche	KK8581
RNA 6000 Nano Kit	Agilent	5067-1511
NEBNext Multiplex Small RNA Library Prep Set for Illumina	New England Biolabs, Inc.	E7300L

Deposited data

Whole genome sequencing (PRJNA859118)	This paper	https://www.ncbi.nlm.nih.gov/
RNA-seq (GSE208300)	This paper	https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE208300
Small RNA-seq (GSE208301)	This paper	https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE208301
Methylation array data (GSE208299)	This paper	https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE208299

Experimental models: Cell lines

hESC lines Lis38 and Lis39	Tel-Aviv Sourasky Medical Center	N/A
hESC lines Lis45 and Lis46	Tel-Aviv Sourasky Medical Center	N/A
hESC line WA09 (H9)	University of Wisconsin	WiCell
hESC line WIBR3	Whitehead Institute for Biomedical Research	N/A
MEF cells – DR4 on ICR background	This paper	N/A

Experimental models: Organisms/strains

Mouse: NSG	Harlan Ltd.	N/A

Oligonucleotides

Primer: ACTIN Forward: CCACGAAACTAC CTTCAACTCC Reverse: GTGATCTCCTTC TGCATCCTGT	This paper	N/A
Primer: STELLA Forward: CGCATGA AAGAAGACCAACAAACAA Reverse: TTAGACACGCAGAAACTGCAGGGA	This paper	N/A
Primer: TFCP2L1 Forward: TCCTTCTTTA GAGGAGAAGC Reverse: ACCAACGTTG ACTGTAATTC	This paper	N/A
Primer: KLF17 Forward: AGCAAGAGATG ACGATTTTC Reverse: GTGGGACATTATT GGGATTC	This paper	N/A
Primer: KLF4 Forward: CGCTCCATTA CCAAGAGCTCAT Reverse: CACGATC GTCTTCCCCTCTTT	This paper	N/A
Primer: KLF5 Forward: CACACTGGTGAAAAGCCATACAA Reverse: GCCTGTGTGCTTCCGGTAGT	This paper	N/A
Primer: NANOG Forward: GCAGAAGGCCTCAGCACCTA Reverse: AGGTTCCCAGTCGGGTTCA	This paper	N/A

Software and algorithms

BD FACSDiva Software	BD Biosciences	https://www.bdbiosciences.com/en-us/products/software/instrument-software/bd-facsdiva-softwa
BlueFuse Multi software	Illumina	https://www.illumina.com/clinical/clinical_informatics/bluefuse.html
FASTQC v.0.11.8	Babraham Bioinformatics	https://www.bioinformatics.babraham.ac.uk/projects/fastqc/
Trim Galore v. 0.4.1	Babraham Bioinformatics	https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/
Bowtie2 v.2.3.4.3	Langmead and Salzberg,¹¹³	https://bowtie-bio.sourceforge.net/bowtie2/index.shtml
Picard Tools v.2.18.15	Broad Institute	https://broadinstitute.github.io/picard/
GATK4 v. 4.0.11.0	Van der Auwera et al.,¹¹⁴	https://gatk.broadinstitute.org/hc/en-us/sections/360007226651-Best-Practices-Workflows
ANNOVAR v. 2019Oct24	Wang et al,¹¹⁵	https://annovar.openbioinformatics.org/en/latest/
vcftools v. 0.1.16	1000 Genomes Project	https://vcftools.github.io/index.html
Bedtools v. 2.25.0	Quinlan Lab University of Utah	https://bedtools.readthedocs.io/en/latest/
CNVkit v. 0.9.6	Talevich et al.,¹¹⁶	https://cnvkit.readthedocs.io/en/stable/
ERDS v. 1.1	Zhu et al.,¹¹⁷	https://github.com/igm-team/ERDS
PURPLE v. 2.34	Priestley et al.,¹¹⁸	https://github.com/hartwigmedical/hmftools/blob/master/purple/README.md
Homer v. 4.10.4	Heinz et al.,¹¹⁹	http://homer.ucsd.edu/homer/
STAR v. 2.7.3a	Dobin et al.,¹²⁰	https://github.com/alexdobin/STAR
featureCounts (subread v. 2.6.3)	Liao et al,¹²¹	https://subread.sourceforge.net/
BiomaRt v. 2.42.1	Ensembl	http://uswest.ensembl.org/info/data/biomart/index.html
DESeq2 v. 1.26.0	Love et al.,¹²²	https://bioconductor.org/packages/release/bioc/html/DESeq2.html
fgsea v. 1.12.0	Korotkevich et al.,¹²³	https://github.com/ctlab/fgsea
Genboree Workbench	Rozowsky et al.,¹²⁴	https://www.genboree.org/site/
Enrichr	Chen et al.,¹²⁵; Kuleshov et al.,¹²⁶; Xie et al.,¹²⁷	https://maayanlab.cloud/Enrichr/
Revigo	Supek et al.,⁶⁰	http://revigo.irb.hr/
CirGO	Kuznetsova et al.,⁶¹	https://github.com/IrinaVKuznetsova/CirGO
Cytoscape v. 3.8.0	Cytoscape Organization	https://cytoscape.org/
stringApp v. 1.5.1	Cytoscape App store	https://apps.cytoscape.org/apps/stringapp
Arboreto v. 0.1.5	Moerman et al.,¹²⁸	https://github.com/aertslab/arboreto
miRPathDB 2.0	Kehl et al.,⁵⁴	https://mpd.bioinf.uni-sb.de/
mirTarBase (release 8.0)	Hsu et al.,¹²⁹; Huang et al.,¹³⁰	https://miRTarBase.cuhk.edu.cn:443/
Scanpy v. 1.3.2	Wolf et al.,¹³¹	https://scanpy.readthedocs.io/en/stable/
Minfi v. 1.32.0	Aryee et al.,¹³²	https://bioconductor.org/packages/release/bioc/html/minfi.html
CpGtools	Wei et al.,¹³³	https://github.com/liguowang/cpgtools

Other

IX51 inverted light microscope	Olympus	https://www.olympus-lifescience.com/en/microscopes/inverted/
Rotor Gene 6000 Series	Corbett	https://www.qiagen.com/us/corbett/welcome/productinfo/
BD FACSCanto II Flow Cytometer	BD Biosciences	https://www.bdbiosciences.com/en-us/products/instruments/flow-cytometers/clinical-cell-analyzers/facscanto
24sure V3 microarray	Illumina	https://www.illumina.com/content/dam/illumina-marketing/documents/products/technotes/24sure-validation-tech-note-1570-2014-026.pdf
Agilent G2565CA scanner	Agilent	https://www.agilent.com/cs/library/usermanuals/Public/G2505-90019_ScannerC_User.pdf
Agilent BioAnalyzer	Agilent	https://www.agilent.com/en/product/automated-electrophoresis/bioanalyzer-systems/bioanalyzer-instrument/2100-bioanalyzer-instrument-228250
NovaSeq S4	Illumina	https://www.illumina.com/systems/sequencing-platforms/novaseq/specifications.html
HiSeq 4000	Illumina	https://www.illumina.com/systems/sequencing-platforms/hiseq-3000-4000/specifications.html
Pippin Prep	Sage Science	https://sagescience.com/products/pippin-prep/#description2d90-f633
Illumina Infinium MethylationEPIC array	Illumina	https://www.illumina.com/products/by-type/microarray-kits/infinium-methylation-epic.html