Transcriptional metabolic reprogramming implements meiotic fate decision in mouse testicular germ cells

. Author manuscript; available in PMC: 2023 Sep 27.

Published in final edited form as: Cell Rep. 2023 Jul 4;42(7):112749. doi: 10.1016/j.celrep.2023.112749

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
PLZF	Santa Cruz	Cat# sc-28319; RRID:AB_2218941
KIT	R&D Systems	Cat# AF1356; RRID:AB_354750
SLC7A5	Cell Signaling Technology	Cat# 5347S; RRID:AB_10695104
SLC38A2	Sigma Aldrich	Cat# HPA035180; RRID:AB_10669901
SLC2A1	Abcam	Cat# ab115730; RRID:AB_10903230
gH2AX	Millpore	Cat# 05–636; RRID:AB_309864
VASA	R&D Systems	Cat# AF2030; RRID:AB_2277369
P70S6K	Cell Signaling Technology	Cat# 9234P; RRID:AB_2269803
p-70S6K	Cell Signaling Technology	Cat# 9202S; RRID:AB_331676
S6	Cell Signaling Technology	Cat# 4858P; RRID:AB_916156
p-S6	Cell Signaling Technology	Cat# 2217S; RRID:AB_331355
4EBP1	Cell Signaling Technology	Cat# 2855P; RRID:AB_560835
p-4EBP1	Cell Signaling Technology	Cat# 9644T; RRID:AB_2097841
FLAG	Sigma Aldrich	Cat# F1804; RRID:AB_262044
a6 integrin	BD Biosciences	Cat# 555736; RRID:AB_396079
KIT	BD Biosciences	Cat# 553356; RRID:AB_398536
donkey anti-mouse IgG Alexa Flour 488	Life Technologies	Cat# A21202; RRID:AB_141607
donkey anti-rabbit IgG Alexa Flour 568	Life Technologies	Cat# A10042; RRID:AB_2534017
donkey anti-goat IgG Alexa Flour 647	Life Technologies	Cat# A21447; RRID:AB_141844
Biological samples
Human testis testicular tissue from organ donors	Center for Organ Recovery and Education	N/A
Chemicals, Peptides, and Recombinant Proteins
Superscript II Reverse Transcriptase	Thermo Fisher Scientific	Cat# 18080051
NuPage 4 %–12 %Bis-Tris Protein Gel	Thermo Fisher Scientific	Cat# NP0322BOX
cOmplete, EDTA-Free	Roche	Cat# 4 693 132
RIPA Buffer	Thermo	Cat# 89900
all-transRA	Sigma-Aldrich	Cat# R2625
2-NBDG	Sigma-Aldrich	Cat# 72987
Glucose-Cy5	Sigma-Aldrich	Cat# SML3233
collagenase IV	Thermo Fisher Scientific	Cat# 17104019
TRIzol	Invitrogen	Cat# 15596018
Power SYBR Green Master Mix	Applied Biosystems	Cat# A25742
Lipofectamine 3000	Invitrogen	Cat# L3000015
N2	R&D Systems	Cat# AR-009
Pyruvic Acid	Sigma-Aldrich	Cat# P2256
D-(+)-glucose	Sigma-Aldrich	Cat# G7021
DL-Lactic Acid	Sigma-Aldrich	Cat# L4263
BSA	MPBiomedicals	Cat# 810661
L-Glutamine	Sigma-Aldrich	Cat# G7513
2-Mercaptoethanol	Sigma-Aldrich	Cat# M3148
MEM Vitamin	Invitrogen	Cat# 11120–052
NEAA	Invitrogen	Cat# 11140–050
Ascorbic Acid	Sigma-Aldrich	Cat# A4544
d-Biotin	Sigma-Aldrich	Cat# B4501
Beta-Estradiol	Sigma-Aldrich	Cat# E2758
FBS	Hyclone	Cat# SH30396.03
KSR	Invitrogen	Cat# 10828–028
Human FGF2	Peprotech	Cat# 100–18B
Rat GDNF	Peprotech	Cat# 450–51
Critical commercial assays
Slc7a5 probe	RNAscope^® Probe	Cat# 461031
Slc38a2 probe	RNAscope^® Probe	Cat# 1184001-C2
Slc2a1 probe	RNAscope^® Probe	Cat# 458671-C2
TruSeq RNA Sample Preparation Kit v2	Illumina	Cat# RS-122–2001
Illumina ChIP-Seq Sample Prep Kit	Illumina	Cat# FC-102–1003
Illumina Sequencing Kit v4	Illumina	Cat# 15003926
Illumina TruSeq ChIP Sample Preparation Kit	Illumina	Cat# IP-202–1012
Chromium Single Cell 3′ v3 Reagent Kits & Chip Kits	10x Genomics	Cat# PN-1000075
Seahorse XF Mito Fuel Flex	Agilent	Cat# 103260–100
Glutamine/glutamate-Glo Assay kit	Promega	J8021
NovaRed Kit	Vector Laboratories	SK-4800
DC protein assay	BioRad	Cat# 5000111
Deposited Data
RNA-Seq of WT, Meiosin KO and Stra8 KO Testes	Ishiguro et al.¹²	DDBJ DRA007066
ChIP-Seq of MEIOSIN	Ishiguro et al.¹²	DDBJ DRA007778
RNA-Seq and ChIP-Seq of GS Cells	Ishiguro et al.¹²	GEO GSE116798
STRA8-FLAG ChIP-Seq Data	Kojima et al.¹¹	GEO GSE115928
Raw and analyzed 10x Genomics scRNA-seq datasets	This paper	GEO GSE211390
In vitro meiosis 10x Genomics scRNA-seq	Zhang et al.¹⁵	GEO GSE153300
Mouse testis Slide-seq	Chen et al.²⁶	PRJNA668433
Experimental models: Cell lines
Cultured mouse spermatogonial stem cells	This paper	N/A
Experimental models: organisms/strains
Mouse: C57BL/6J	Charles River Laboratory	Strain code: 027
Mouse: Stra8Knockout	The Jackson Laboratory	JAX:023805
MEF	R&D systems	Cat# PSC001
Oligonucleotides
Slc7a5_siRNA	Dharmacon	Cat# 041166-01-0005
Slc38a2 siRNA	Dharmacon	Cat# 059380-00-0005
Slc2a1_siRNA	Dharmacon	Cat# 044254-02-0005
Ctrl siRNA	Dharmacon	Cat# D-001210-01-05
Primers for qRT-PCR	This paper	Table S1
Recombinant DNA
Stra8-flag	This paper	N/A
Stra8^Δ1	This paper	N/A
Stra8^Δ2	This paper	N/A
Stra8^Δ3	This paper	N/A
Stra8^ΔC	This paper	N/A
Software and algorithms
FlowJo v10	FlowJo, LLC	https://www.flowjo.com/
Cell Ranger v5.1.0	10x Genomics	https://support.10xgenomics.com/single-cell-gene-expression/software/downloads/latest
Seurat R package Version 3.0	Butler et al.⁴²	https://satijalab.org/seurat/
R Version 3.4 or higher	N/A	https://www.r-project.org
R studio Version 1.0.143	N/A	https://www.rstudio.com
GraphPad Prism (v9.0)	N/A	https://www.graphpad.com/scientific-software/prism/
ImageJ v1.41	NIH	https://imagej.net/ij/
DropletUtils	Griffiths et al.⁴³	https://www.nature.com/articles/s41467-018-05083-x
DoubletFinder	McGinnis et al.⁴⁴	https://www.sciencedirect.com/science/article/pii/S2405471219300730
Harmony	Korsunsky et al.⁴⁵	https://www.nature.com/articles/s41592-019-0619-0
Pheatmap	Kolde.⁴⁶	https://cran.r-project.org/web/packages/pheatmap/index.html
ClusterProfiler	Yu et al.⁴⁷	https://www.liebertpub.com/doi/10.1089/omi.2011.0118?url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org&rfr_dat=cr_pub++0pubmed&
AlphaFold2	Jumper et al.⁴⁸	https://www.nature.com/articles/s41586-021-03819-2
MMseqs2	Steinegger et al.⁴⁹	https://www.nature.com/articles/nbt.3988
PyMOL	DeLano et al.⁵⁰	https://pymol.org/2/