Variant-to-function analysis of the childhood obesity chr12q13 locus implicates rs7132908 as a causal variant within the 3′ UTR of FAIM2

. 2024 May 1;4(5):100556. doi: 10.1016/j.xgen.2024.100556

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

Anti-MAP2 (Chicken, Polyclonal)	Abcam	Cat# ab5392; Lot# GR3450786-1; RRID: AB_2138153
Anti-TTF-1/NKX2.1 (Mouse, Monoclonal)	Cell Marque	Cat# 343M-95; Lot# 0000051910; RRID: AB_1158934
Anti-NeuN (Mouse, Monoclonal)	Millipore Sigma	Cat# MAB377; RRID: AB_2298772
Anti-Chicken Alexa Fluor 488 (Goat, Polyclonal)	Abcam	Cat# ab150169; RRID: AB_2636803
Anti-Mouse Alexa Fluor 488 (Goat, Polyclonal)	Invitrogen	Cat# A-11001; RRID: AB_2534069

Biological samples

Frozen hypothalamus region, left hemisphere. 1674, 8 years old, male	NIH NeuroBioBank	N/A
Frozen hypothalamus region, left hemisphere. 5309, 14 years old, female	NIH NeuroBioBank	N/A
Frozen hypothalamus region, left hemisphere. 5976, 4 years old, female	NIH NeuroBioBank	N/A
Frozen hypothalamus region, left hemisphere. 6032, 4 years old, male	NIH NeuroBioBank	N/A

Chemicals, peptides, and recombinant proteins

1M HEPES	Gibco	Cat# 15630-080
0.25% Trypsin-EDTA	Gibco	Cat# 25200-056
Dulbecco’s Phosphate-Buffered Saline (DPBS) without calcium and magnesium	Corning	Cat# 21-031-CV
Heat-Inactivated FBS	Gibco	Cat# 10082-147
Dulbecco’s Modified Eagle’s Medium (DMEM)	ATCC	Cat# 30-2002
100X Antibiotic-Antimycotic	Gibco	Cat# 15240062
200 mM L-Glutamine	Corning	Cat# 25-005-CI
PBS, pH 7.4	Gibco	Cat# 10010023
ROCK Inhibitor Y-27632	Stemcell Technologies	Cat# 72304
Matrigel hESC-Qualified Matrix, LDEV-free	Corning	Cat# 354277
Versene Solution	Gibco	Cat# 15040-066
DMSO	Sigma	Cat# D2650-100ML
mFreSR	Stemcell Technologies	Cat# 05854
Accutase	Stemcell Technologies	Cat# 07920
Opti-MEM Reduced Serum Media	Gibco	Cat# 31985-062
4% Paraformaldehyde	Biotium	Cat# 22023
XhoI	NEB	Cat# R0146S
Miller’s LB Broth	Corning	Cat# 46-050-CM
LB + Ampicillin (100 μg/mL) Agar Plates	University of Pennsylvania Cell Center Service Facility	Cat# 6005
Ampicillin	Corning	Cat#61-238-RH
AflII	NEB	Cat# R0520S
Lipofectamine Stem Transfection Reagent	Invitrogen	Cat# STEM00003
BfaI	NEB	Cat# R0568S
TRIzol Reagent	Invitrogen	Cat# 15596018
DNase I	Zymo	Cat# E1009-A
100% Ethanol	Electron Microscopy Sciences	Cat#15055
TrypLE Express Enzyme	ThermoFisher	Cat# 12605036
DMEM/F12	Gibco	Cat# 11320-033
Matrigel Growth Factor Reduced Basement Membrane Matrix	Corning	Cat# 354230
Iscove’s Dulbecco’s Medium (IMDM)	Gibco	Cat# 12440053
KnockOut Serum Replacement	Gibco	Cat# 10828-010
MEM Non-Essential Amino Acids Solution	Gibco	Cat# 11140050
100 mM Sodium Pyruvate	Gibco	Cat# 11360070
Penicillin-Streptomycin (10,000 U/mL)	Gibco	Cat# 15140122
β-Mercaptoethanol	Sigma Life Science	Cat# M3148-25ML
Recombinant Human FGF basic/FGF2/bFGF (146 aa) Protein	R&D Systems	Cat# 233-FB
1M Tris-HCl Buffer, pH 7.5	Invitrogen	Cat# 15567027
Tween 20, 10% (w/v)	Roche	Cat# 11332465001
JumpStart Taq DNA Polymerase	Sigma Aldrich	Cat# D6558-50UN
AMPure XP Beads	Beckman Coulter	Cat# A63881
IGEPAL CA-630	Sigma	Cat# I8896-50ML
Triton X-100	Sigma	Cat# X100-5ML
10X PBST Buffer Strength Solution	Bio Basic	Cat# PW004
DAPI (4′,6-Diamidino-2-Phenylindole, Dilactate)	Invitrogen	Cat# D3571
ProLong Gold Antifade Mountant	Invitrogen	Cat# P36930
Corticosterone	Sigma	Cat# 27840
Linoleic Acid	Sigma	Cat# L1376
Linolenic Acid	Sigma	Cat# L2376
(±)-α-Lipoic Acid	Sigma	Cat# T5625
Progesterone	Sigma	Cat# P0130
Retinyl Acetate	Sigma	Cat# 46958
(±)-α-Tocopherol	Sigma	Cat# T3251
DL-α-Tocopherol Acetate	Sigma	Cat# T3376
Bovine Serum Albumin	Sigma	Cat# A4919
Sodium Bicarbonate	Sigma	Cat# S5761
L-Ascorbic Acid	Sigma	Cat# A8960
Putrescine Dihydrochloride	Sigma	Cat# P5780
D(+)-Galactose	Sigma	Cat# G5388
Holo-Transferrin	Sigma	Cat# T0665
Catalase	Sigma	Cat# C1345
L-Carnitine Hydrochloride	Sigma	Cat# C0283
Glutathione	Sigma	Cat# G4251
Sodium Selenite	Sigma	Cat# S5261
Ethanolamine	Sigma	Cat# E9508
Triiodo-L-Thyronine Sodium Salt	Sigma	Cat# T6397
Insulin Solution, Human	Sigma	Cat# 19278
Superoxide Dismutase	Sigma	Cat# S5395
LDN-193189 (hydrochloride)	Cayman Chemical	Cat# 19396
SB-431542 (hydrate)	Cayman Chemical	Cat# 13031
SAG	Cayman Chemical	Cat# 11914
Purmorphamine	Tocris	Cat# 4551
IWR-1-Endo	Cayman Chemical	Cat# 13659
DAPT	Cayman Chemical	Cat# 13197
All-Trans Retinoic Acid	Cayman Chemical	Cat# 11017
Human BDNF	Miltenyi Biotec	Cat# 130-093-811
Laminin	Sigma	Cat# L2020-1MG
Hanks' Balanced Salt Solution	Sigma	Cat# H8264-100ML
7.5% Bovine Serum Albumin Solution	Sigma	Cat# A8412-100ML
1M Trizma Hydrochloride, pH 7.4	Sigma	Cat# T2194-100ML
5M Sodium Chloride Solution	Sigma	Cat# 59222C-500ML
1M Magneisum Chloride Solution	Sigma	Cat# M1028-100ML
Protector RNase Inhibitor	Roche	Cat# 03335402001
100 mM DTT	Agilent	Cat# 600089-53
GlutaMAX	Gibco	Cat# 35050061

Critical commercial assays

AGM Astrocyte Growth Medium BulletKit	Lonza	Cat# CC-3186
mTeSR1 Complete Kit	Stemcell Technologies	Cat# 85850
LookOut Mycoplasma PCR Detection Kit	Sigma Aldrich	Cat# MP0035-1KT
Lipofectamine LTX Reagent with PLUS Reagent	Invitrogen	Cat# 15338030
Monarch DNA Gel Extraction Kit	NEB	Cat# T1020S
Gibson Assembly HiFi HC 1-Step Kit	Codex	Cat# GA1100-4X10
NEB Stable Competent E. coli (High Efficiency)	NEB	Cat# C3040H
QIAprep Spin Miniprep Kit	Qiagen	Cat# 27106
EndoFree Plasmid Maxi Kit	Qiagen	Cat# 12362
Q5 Site-Directed Mutagenesis Kit	NEB	Cat# E0552S
NEBNext High-Fidelity 2X PCR Master Mix	NEB	Cat# M0541S
Lipofectamine 3000 Transfection Reagent	Invitrogen	Cat# L3000001
Dual-Luciferase Reporter Assay System	Promega	Cat# E1960
Direct-zol RNA Miniprep Kit	Zymo	Cat# R2050
Qubit RNA High Sensitivity Assay Kit	Invitrogen	Cat# Q32855
SuperScript IV VILO Master Mix with ezDNase Enzyme	Invitrogen	Cat# 11766050
Phusion High-Fidelity DNA Polymerase	NEB	Cat# M0530S
NucleoSpin Gel and PCR Clean-Up Kit	Takara	Cat# 740609
Gibson Assembly Kit	NEB	Cat# E2611
FAIM2 TaqMan Gene Expression Assay	ThermoFisher Scientific	Cat# 4331182, Assay ID Hs00392345_m1
18S TaqMan Gene Expression Assay	ThermoFisher Scientific	Cat# 4331182, Assay ID Hs99999901_s1
TaqMan Fast Advanced Master Mix	Applied Biosystems	Cat# 4444557
Quick-DNA Miniprep Plus Kit	Zymo	Cat# D4068
Infinium Global Screening Array-24 v3.0 Kit	Illumina	Cat# 20030770
Infinium OmniExpressExome-8 v1.6 Kit	Illumina	Cat# 20024676
Quick-DNA/RNA Miniprep Plus Kit	Zymo	Cat# D7003
RNA 6000 Nano Kit	Agilent	Cat# 5067-1511
QIAseq FastSelect RNA Removal Kit	Qiagen	Cat# 333180
NEBNext Ultra II Directional RNA Library Prep for Illumina Kit	NEB	Cat# E7760S
NEBNext Oligos for Illumina (Dual Index Primers Set 1)	NEB	Cat# E7600S
Qubit dsDNA High Sensitivity Assay Kit	Invitrogen	Cat# Q32851
DNA 1000 Kit	Agilent	Cat# 5067-1504
Tagment DNA TDE1 Enzyme and Buffer Kit	Illumina	Cat# 20034197
Nextera DNA CD Indexes Kit	Illumina	Cat# 20018708
MinElute PCR Purification Kit	Qiagen	Cat# 28004
High Sensitivity D1000 ScreenTape Assay	Agilent	Cat# 5067–5587, 5067–5585, 5067–5603, 5067-5584
High Sensitivity D5000 ScreenTape Assay	Agilent	Cat# 5067–5594, 5067–5593, 5067-5592
Chromium Next GEM Chip J Single Cell Kit	10X Genomics	Cat# 1000230
Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle	10X Genomics	Cat# 1000285
Dual Index Kit TT Set A	10X Genomics	Cat# 1000215
Arima-HiC Kit	Arima Genomics	Cat# A510008
Swift Accel-NGS 2S Plus DNA Library Kit	Swift Biosciences	Cat# 21024/21096
Swift 2S Indexing Kit	Swift Biosciences	Cat# 28096
KAPA Library Quantification Kit	Roche	Cat# 07960140001

Deposited data

Raw and processed Hi-C data	This paper	GEO: GSE241592; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE241592
Raw and processed RNA-seq data	This paper	GEO: GSE241050; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE241050
Raw and processed ATAC-seq data	This paper	GEO: GSE241591; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE241591
Raw and processed single-nucleus RNA-seq data	This paper	GEO: GSE241594; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE241594
Raw and processed single-nucleus ATAC-seq data	This paper	GEO: GSE241593; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE241593

Experimental models: Cell lines

NHA - Human Astrocytes	Lonza	Cat# CC-2565
HEK293T Cells	ATCC	Cat# CRL-3216; RRID: CVCL_0063
WA09 (H9) Human Embryonic Stem Cells (NIH Approval Number: NIHhESC-10-0062)	WiCell Research Institute	Lot# DL-05; RRID: CVCL_9773
WA09 (H9) Human Embryonic Stem Cells (NIH Approval Number: NIHhESC-10-0062), rs7132908 AA Clone 2.1	This paper	N/A
WA09 (H9) Human Embryonic Stem Cells (NIH Approval Number: NIHhESC-10-0062), rs7132908 AA Clone 9.1	This paper	N/A
WA09 (H9) Human Embryonic Stem Cells (NIH Approval Number: NIHhESC-10-0062), rs7132908 AA Clone 10.1	This paper	N/A

Oligonucleotides

Oligonucleotides	See Table S13	N/A

Recombinant DNA

LentiCRISPRv2-mCherry plasmid	Agata Smogorzewska	Addgene Cat# 99154; RRID: Addgene_99154
FAIM2 miRNA 3′ UTR target clone in pEZX-MT05 reporter vector	GeneCopoeia	Cat# HmiT096491-MT05
FAIM2 promoter clone in pEZX-PG02 reporter vector	GeneCopoeia	Cat# HPRM47354-PG02
LIMA1 promoter clone in pEZX-PG02 reporter vector	GeneCopoeia	Cat# HPRM34453-PG02
RACGAP1 promoter clone in pEZX-PG02 reporter vector	GeneCopoeia	Cat# HPRM34625-PG02
pGL4.10[luc2] reporter vector	Promega	Cat# E6651
pRL-TK reporter vector	Promega	Cat# E2241
pGL4.10[luc2]-rs7132908G-FAIM2	This paper	N/A
pGL4.10[luc2]-rs7132908A-FAIM2	This paper	N/A
pGL4.10[luc2]-FAIM2	This paper	N/A
pGL4.10[luc2]-rs7132908G-LIMA1	This paper	N/A
pGL4.10[luc2]-rs7132908A-LIMA1	This paper	N/A
pGL4.10[luc2]-LIMA1	This paper	N/A
pGL4.10[luc2]-rs7132908G-RACGAP1	This paper	N/A
pGL4.10[luc2]-rs7132908A-RACGAP1	This paper	N/A
pGL4.10[luc2]-RACGAP1	This paper	N/A
gRNA_Cloning vector	Mali et al.⁸³	Addgene Cat# 41824; RRID: Addgene_41824
gRNA_Cloning-rs7132908gRNA vector	This paper	N/A
pCas9_GFP	Ding et al.⁸⁴	Addgene Cat# 44719; RRID: Addgene_44719

Software and algorithms

SnapGene v6.0.2	SnapGene	https://www.snapgene.com/; RRID: SCR_015052
Prism v10.0.0	GraphPad	https://www.graphpad.com/features; RRID: SCR_002798
CRISPOR v5.01	Concordet⁸⁵	http://crispor.tefor.net/; RRID: SCR_015935
Excel v2202	Microsoft	RRID: SCR_016137
AriaMx v1.5	Agilent	https://www.agilent.com/en/product/real-time-pcr-%28qpcr%29/real-time-pcr-%28qpcr%29-instruments/ariamx-software-download
2100 Bioanalyzer Expert vB.02.11.SI824	Agilent	https://explore.agilent.com/Software-Download-2100-Expert?productURL=https%3A%2F%2Fwww.agilent.com%2Fen%2Fproduct%2Fautomated-electrophoresis%2Fbioanalyzer-systems%2Fbioanalyzer-software%2F2100-expert-software-228259; RRID: SCR_019715
FastQC v0.11.9	Andrews⁸⁶; FASTQC⁸⁷	https://www.bioinformatics.babraham.ac.uk/projects/fastqc/; RRID: SCR_014583
Kallisto v0.48.0	Bray et al.⁸⁸; Bray et al.⁸⁹	https://pachterlab.github.io/kallisto/; RRID: SCR_016582
bcl2fastq2 Conversion v2.20	Illumina	https://sapac.support.illumina.com/downloads/bcl2fastq-conversion-software-v2-20.html; RRID: SCR_015058
R v4.2.2 and v4.2.3	Comprehensive R Archive Network	https://cran.r-project.org/; RRID: SCR_001905
RStudio v2022.07 and v2023.06.0 + 421	Posit	https://posit.co/download/rstudio-desktop/
cellSens Standard v2.3	Olympus	https://www.olympus-lifescience.com/en/software/cellsens/
ImageJ v1.54days	Schneider et al.⁹⁰	https://imagej.nih.gov/ij/download.html; RRID: SCR_003070
PennCNV v1.0.5	GitHub	https://penncnv.openbioinformatics.org/en/latest/user-guide/download/; RRID: SCR_002518
PLINK v1.90b6.18	Harvard University	https://zzz.bwh.harvard.edu/plink/download.shtml; RRID: SCR_001757
Cell Ranger ARC v2.0.2	10X Genomics	https://support.10xgenomics.com/single-cell-multiome-atac-gex/software/pipelines/latest/installation; RRID: SCR_023897
Scrublet v0.2.3	Wolock et al.⁹¹	https://github.com/swolock/scrublet; RRID: SCR_018098
SoupX v1.6.2	Young et al.⁹²	https://github.com/constantAmateur/SoupX; RRID: SCR_019193
Harmony v0.1.1	Korsunsky et al.⁹³	https://cran.r-project.org/web/packages/harmony/index.html; RRID: SCR_022206
TapeStation Analysis Software v4.1.1	Agilent	https://www.agilent.com/en/product/automated-electrophoresis/tapestation-systems/tapestation-software/tapestation-software-379381
Seurat v4.3.0	Hao et al.⁹⁴	https://satijalab.org/seurat/articles/install.html; RRID: SCR_016341
SCTransform v0.3.5	Choudhary et al.⁹⁵; Hafemeister et al.⁹⁶	https://cran.r-project.org/web/packages/sctransform/index.html; RRID: SCR_022146
gprofiler2 v0.2.1	Raudvere et al.⁹⁷; Kolberg et al.⁹⁸	https://cran.r-project.org/web/packages/gprofiler2/vignettes/gprofiler2.html; RRID: SCR_018190
tximport v1.24.0	Soneson et al.⁹⁹	https://bioconductor.org/packages/release/bioc/html/tximport.html; RRID: SCR_016752
edgeR v3.38.4 and v3.40.2	Robinson et al.¹⁰⁰	https://bioconductor.org/packages/release/bioc/html/edgeR.html; RRID: SCR_012802
ensembldb v2.20.2	Rainer et al.¹⁰¹	https://bioconductor.org/packages/release/bioc/html/ensembldb.html; RRID: SCR_019103
EnsDb.Hsapiens.v86 v2.99.0	Bioconductor	https://bioconductor.org/packages/release/data/annotation/html/EnsDb.Hsapiens.v86.html
limma v3.52.4	Ritchie et al.¹⁰²	https://bioconductor.org/packages/release/bioc/html/limma.html; RRID: SCR_010943
bowtie2 v2.2.6	Langmead et al.¹⁰³	https://bowtie-bio.sourceforge.net/bowtie2/index.shtml; RRID: SCR_016368
Picard v2.7.1	GitHub	https://github.com/broadinstitute/picard; RRID: SCR_006525
SAMtools v1.7	Danecek et al.¹⁰⁴	http://www.htslib.org/; RRID: SCR_002105
MACS2 v2.1.1	Zhang et al.¹⁰⁵	https://pypi.org/project/MACS2/
csaw v1.32.0	Lun et al.¹⁰⁶	https://bioconductor.org/packages/release/bioc/html/csaw.html
ggplot2 v3.4.2	Wickham¹⁰⁷	https://cran.r-project.org/web/packages/ggplot2/index.html; RRID: SCR_014601
pheatmap v1.0.12	Comprehensive R Archive Network	https://cran.r-project.org/web/packages/pheatmap/index.html; RRID: SCR_016418
plotly v4.10.1	Comprehensive R Archive Network	https://cran.r-project.org/web/packages/plotly/index.html
MACS3 v3.0.0b2	GitHub	https://github.com/macs3-project/MACS
Signac v1.10.0	Stuart et al.¹⁰⁸	https://stuartlab.org/signac/articles/install.html; RRID: SCR_021158
DESeq2 v1.38.3	Love et al.¹⁰⁹	https://bioconductor.org/packages/release/bioc/html/DESeq2.html; RRID: SCR_015687
SingleCellExperiment v1.20.1	Amezquita et al.¹¹⁰; Amezquita et al.¹¹¹	https://bioconductor.org/packages/release/bioc/html/SingleCellExperiment.html
Matrix.utils v0.9.8	Comprehensive R Archive Network	https://rdrr.io/cran/Matrix.utils/
apeglm v1.20.0	Zhu et al.¹¹²	https://bioconductor.org/packages/release/bioc/html/apeglm.html
gplots v3.1.3	Comprehensive R Archive Network	https://cran.r-project.org/web/packages/gplots/index.html
UCSC Genome Browser (GRCh37/hg19)	University of California Santa Cruz	https://genome.ucsc.edu/cgi-bin/hgGateway; RRID: SCR_005780
HICUP pipeline v0.7.4	Wingett et al.¹¹³	https://www.bioinformatics.babraham.ac.uk/projects/hicup/; RRID: SCR_005569
Pairtools v0.3.0	Open2C et al.¹¹⁴	https://pairtools.readthedocs.io/en/latest/installation.html; RRID: SCR_023038
Pairix v0.3.7	Lee et al.¹¹⁵	https://github.com/4dn-dcic/pairix
Cooler v0.8.11	Abdennur et al.¹¹⁶	https://pypi.org/project/cooler/
Mustache v1.0.1	Roayaei Ardakany et al.¹¹⁷	https://github.com/ay-lab/mustache
Fit-Hi-C2 v2.0.7	Kaul et al.¹¹⁸	https://github.com/ay-lab/fithic
BSgenome v1.68.0	Bioconductor	https://bioconductor.org/packages/release/bioc/html/BSgenome.html
SNPlocs.Hsapiens.dbSNP155.GRCh38 0.99.24	Bioconductor	https://bioconductor.org/packages/release/data/annotation/html/SNPlocs.Hsapiens.dbSNP155.GRCh38.html
MotifDb v1.42.0	Bioconductor	https://bioconductor.org/packages/release/bioc/html/MotifDb.html
motifbreakR v2.14.2	Coetzee et al.¹¹⁹	https://bioconductor.org/packages/release/bioc/html/motifbreakR.html
ColoQuiaL	Chen et al.¹²⁰	https://github.com/bvoightlab/ColocQuiaL
STAR v2.7.9a	Dobin et al.¹²¹	https://github.com/alexdobin/STAR; RRID: SCR_004463
HTSeq-count v0.11.3	Anders et al.¹²²	https://shicheng-guo.github.io/research/1941/01/08/HTseq; RRID: SCR_011867
SKAT v2.2.5	Comprehensive R Archive Network	http://cran.nexr.com/web/packages/SKAT/index.html; RRID: SCR_009396
PVCA v3.18	Bioconductor	https://www.bioconductor.org/packages/release/bioc/html/pvca.html; RRID: SCR_001356

Other

Falcon Round-Bottom Polystyrene Test Tubes with Cell Strainer Snap Cap, 35 μm, 5 mL	Fisher Scientific	Cat# 08-771-23
ZR BashingBead Lysis Tubes, 2 mm	Zymo	Cat# 56003-50
#1.5 Acid-Treated Coverslips, 12 mm diameter	Fisher Scientific	Cat# NC0706236
Nunc Cell-Culture Treated 6-well Plates	ThermoFisher Scientific	Cat# 140675
Costar 24-well Clear TC-Treated Multiple Well Plates	Corning	Cat# 3524
75 cm2 U-Shaped Canted Neck Cell Culture Flask with Plug Seal Cap	Corning	Cat# 430720U
Falcon 25 cm2 Rectangular Canted Neck Cell Culture Flask with Blue Plug Seal Screw Cap	Corning	Cat# 353014
Falcon 100 mm TC-Treated Cell Culture Dish	Corning	Cat# 353003
White 96-well Immuno Microlite 1+ Plates	Thermo Scientific	Cat# 7571