PMCID string | Title string | Sentences string |
|---|---|---|
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The most likely imputed genotypes were selected for statistical analysis if the highest probability (r2) > 0.9. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | SNP markers showing deviation from Hardy–Weinberg equilibrium in European controls, a minor allele frequency or MAF <5%, or imputation INFO scores <0.5 were filtered out of all subsequent analyses. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The information for the genotyped markers was retained after imputation, and the imputed values for these variants were only used to assess concordance. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Our methods for meta-analysis were previously published. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Case-parent trios were analyzed with the allelic transmission disequilibrium test (TDT) implemented in PLINK. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Unrelated cases and controls, matched for population of origin, were tested for association using a logistic regression under an additive genetic model while including 18 principal components of ancestry to adjust for population structure. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Within each OFC subtype, the resulting effect estimates for the TDT and logistic regression were combined in an inverse-variance weighted fixed-effects meta-analysis. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We selected 887 SNPs from eight loci that include a gene known to be expressed in oral epithelium and in most cases, regulates differentiation of an epithelial tissue (1q32/IRF6, 2p21/THADA, 6p24.3/TFAP2A, 9q22.2/GADD45G, 12q13.13/KRT18, 20q12/MAFB and 9q22.33/FOXE1) (Table 1 and Supplementary Data 1, 2). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We selected SNPs with P values suggestive of association from seven loci (IRF6, THADA, TFAP2A, GADD45G, KRT18, and MAFB) from the meta-analysis (Table 1 and Supplementary Data 2). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We additionally included 59 SNPs at the TFAP2A locus identified in an independent GWAS of CL/P in Han Chinese. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Because of the financial constraints on the library size, for one locus (FOXE1) we instead picked nine SNPs in strong linkage disequilibrium with the lead SNP (rs12347191) in a European population and annotated by Haploreg as being within regulatory elements (Supplementary Data 2). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We designed an oligonucleotide library in which each oligo contained the sequence of the SNP of interest, a unique barcode, and priming and restriction sites necessary for amplification and cloning into a pGL-hsp68 reporter plasmid. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The sequence of human genomic DNA corresponding to 161 bp windows, centered on each of 887 GWAS-identified SNPs, was downloaded from the UCSC Table browser tool (genome build: hg19). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Two additional SNPs, absent from all tables, supplementary data and schematics, were included in the MPRA as part of a separate preliminary study. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We generated two versions of each cis-regulatory element (CRE) that were identical except for harboring the major or minor allele of the SNP, respectively (1774 elements) (Fig. 1a and Supplementary Data 3). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We ordered a pool of 7500 unique 230-mer oligonucleotides (elements) through a limited licensing agreement with Agilent Technologies (Santa Clara, CA). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | To establish an empirical null distribution of activity in the assay, we included 90 sequences that were scrambled versions of the sequences of other elements (Supplementary Data 4). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | To account for noise in the assay, each sequence was assigned four unique barcodes. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Finally, to create constructs with only the basal promoter and no upstream regulatory sequence, we included an additional random 28 filler elements, which were excised in the second step of cloning, yielding 28 basal promoter-only controls with one barcode each. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We synthesized 7484 elements corresponding to the 887 SNPs (7096 + 360 + 28) (Supplementary Data 4 and Fig. 1a). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Oligos were designed with the following structure (Supplementary Fig. 21): a forward priming sequence (PS1) with NheI restriction site, (primer for this site is KC_1F), 161 base cis-regulatory element (Supplementary Data 3), AgeI restriction site, NruI restriction site, MluI restriction site, 9 base barcode, and a reverse priming sequence (PS2) with XhoI site (primer for this site is KC_1R) (Fig. 1a, Supplementary Fig. 21, and Supplementary Data 4). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We amplified the oligo pool using five cycles of PCR with Phusion High-Fidelity polymerase (New England Biolabs) and primer set KC_1 (Supplementary Data 23). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We cloned the pool of amplicons into the pGL-hsp68 backbone using NheI and XhoI, which excludes the hsp68 promoter fused to DsRed. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We prepared DNA from 41,000 colonies to generate library KC_L1. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We then amplified a DNA fragment containing the hsp68 promoter and the DsRed cDNA using primers KC_2F and KC_2R (Supplementary Data 23) containing AgeI and MluI restriction sites, respectively, and cloned this fragment into the KC_L1 library such that the CREs lie upstream of the hsp68 promoter driving DsRed and the barcodes; this procedure created library KC_L2. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | To assess the completeness of the cloned library, we sequenced the barcodes in the final plasmid pool in library KC_L2. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | It was achieved by amplifying the barcode fragments using primer set KC_BC containing with NheI and MfeI restriction sites, followed by specific Illumina adapters ligation (P1 and PE2 adapters), and Illumina enrichment PCR using KC_3 set of primers, and sequencing (Supplementary Data 23). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The human fetal oral epithelial cell line (GMSM-K) (a kind gift from Dr. Daniel Grenier, Université Laval) was maintained in keratinocyte serum-free medium (KSFM) (Gibco, Catalog no. 17005-042) supplemented with human recombinant epidermal growth factor (EGF 30 µg/ml) and bovine pituitary extract (BPE 0.2 ng/ml). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Human Epidermal Keratinocytes from neonatal foreskin (HEKn; primary neonatal keratinocytes) purchased from ATCC (ATCC Catalog no. PCS-200-010) were maintained and cultured as per the manufacturer’s instructions in dermal cell basal medium (ATCC Catalog no. PCS-200-030) supplemented with keratinocyte growth kit (ATCC, Catalog no. PCS-200-040). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The Normal Human Skin Keratinocyte (N-HSK-1) cell line was grown in KSFM or on an irradiated NIH3T3 feeder layer in Dulbecco’s Modified Eagle’s Medium (DMEM) and HamF12 (3:1 ratio) according to Rheinwald and Green. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | WTC-11 human induced pluripotent stem cells (iPSCs) (Coriell Institute for Medical Research, Catalog no. GM25256), were maintained in mTeSR Plus media (StemCell Technologies, Catalog no. 100-0276) on Matrigel (Corning, Catalog no. 356231)-coated plates and passaged with StemPro Accutase (Gibco, Catalog no. A11105-01). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Cells were incubated at 37 °C in 5% CO2. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Details of cell lines/primary cells/induced pluripotent cells used in the study are provided in Supplementary Data 5. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Sex was not considered in tests of allele-specific effects of SNPs on enhancer activity. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | A pool of plasmids (from library KC_L2; as described in Methods, Reporter library construction) in four replicates, 2.5 µg each, was transfected into 1 million GMSM-K with Amaxa Cell Line Nucleofector Kit V (Lonza, Catalog no. VCA-1003) using Nucleofector II (Lonza) (program T-020). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Forty-eight hours post-transfection, RNA and genomic DNA was extracted from each set using Quick-DNA/RNA Miniprep kit (Zymo Research, Catalog no. D7001) as per the manufacturer’s instructions. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Isolated RNA was treated with Turbo DNase I (Thermo Fisher, Catalog no. AM1907) as per the manufacturer’s instructions. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The first strand of cDNA was synthesized with SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific, Catalog no. 18080051). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The barcodes including the flanking sequence were amplified from both cDNA, genomic DNA (transfected plasmid DNA) and plasmid DNA samples (that was used for transfection) with Phusion High-Fidelity PCR Master Mix (NEB, Catalog no. M0531S) using primer set KC_BC (Supplementary Data 23) and that yielded barcode sequences that were flanked with NheI and MfeI restriction enzyme sites. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The products were digested with NheI and MfeI and then ligated with Illumina adapter sequences P1 and PE2 (Supplementary Data 23). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | This product was further amplified using primer set KC_3 (Supplementary Data 23), followed by sequencing. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | To measure expression of the barcoded library, electroporation replicates were multiplexed and run on two lanes of an Illumina HiSeq machine, which generated 48.2 million sequence reads corresponding to cDNA and 48.8 million reads corresponding to DNA (from plasmids). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Sequencing reads that matched the first 20 nucleotides of the designed sequence were counted, regardless of quality score. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We filtered out barcodes that were low abundance in the plasmid pool (<50 reads), then set any cDNA barcodes that were detection-limited in at least one sample (<50 reads for replicates run on the first lane, <100 reads for the replicates run on the second lane) to zero across all cDNA samples. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We normalized each sample to the total counts per million (CPM). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | RNA barcode levels were highly correlated among four replicates (Supplementary Fig. 22). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | There was a strong correlation in the barcode counts in the plasmid library before transfection and after harvesting plasmid from transfected cells (Supplementary Fig. 23). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Therefore, we used plasmid barcode counts (pre-transfection) for further analyses. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We further normalized RNA barcode counts by the plasmid barcode counts (RNA/DNA). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We checked the reproducibility based on log2RNA/DNA counts (Supplementary Fig. 24). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | To calculate the reporter activity for each sequence we averaged the log2RNA/DNA counts within a replicate, normalized to the within-replicate mean basal barcode activity and then averaged across all the replicates (Supplementary Fig. 1a). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The standard error of the mean (SEM) was calculated using the basal-normalized activities across replicates. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Variant significance was determined by performing Welch’s t-test between major and minor alleles, followed by Benjamini–Hochberg false discovery rate (FDR) correction (Supplementary Data 6). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We used our previously published Python code for processing and analyzing MPRA reads, which is available on Github at [https://github.com/barakcohenlab/CRX-Information-Content]. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The activity-by-contact method (ABC) utilizes cell type-specific chromatin accessibility (ATAC-seq or Dnase hypersensitivity) and chromatin activity (H3K27ac) and HiC data that is either cell-type specific or averaged over ten cell types—the performance of this tool is similar with either type of HiC data. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | To calculate the ABC scores for keratinocyte enhancers we used publicly available datasets from primary Normal Human Epidermal Keratinocytes (NHEK) cells for H3K27ac (NCBI GEO Identifier GSM733674), Dnase hypersensitivity (NCBI GEO Identifier GSM736545), RNA-seq (NCBI GEO Identifier GSM958736) and averaged HiC data from 10 cell types as reported in ref. . |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | All elements with an ABC score >0.01 were included in the analysis. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | For dual luciferase reporter assays, each reporter construct (Supplementary Data 10) was cloned into the pGL3 promoter vector, and constructs were co-transfected with Renilla luciferase plasmid. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Briefly, GMSM-K or HEKn cells were electroporated with plasmid using the Amaxa Cell Line Nucleofector Kit V (Lonza, Catalog no. VCA-1003) and the Nucleofector II instrument (Lonza) program T-020. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We used a dual luciferase reporter assay system (Promega, Catalog no. E1980) and FB12 Luminometer (Berthold Detection Systems) to evaluate the luciferase activity following the manufacturer’s instructions. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Relative luciferase activity was the ratio of Firefly and Renilla activities. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | At least three independent measurements were performed for each transfection group. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | All results were presented as mean ± standard deviation (SD). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Statistical significance was determined using a two-tailed Student’s t-test. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | GMSM-K and normal human skin keratinocytes (N-HSK-1) were grown on glass coverslips, fixed in 4% paraformaldehyde for 10 min, permeabilized in 0.2% Triton X-100 for 2 min, then rinsed in PBS. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Cells were incubated in 3% goat serum (Vector Laboratories, Catalog no. S-1000-20) in 2% PBS-bovine serum albumin for 30 min to block non-specific binding, followed by incubation in primary then secondary fluorescent antibodies 1 h each at room temperature with PBS washes in between the two incubations. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Primary antibodies that were used are: mouse monoclonal anti-E-cadherin (BD Bioscience, Catalog no. 610181, 1/100), mouse monoclonal anti P63 (clone 4A4, Santa Cruz, Catalog no. SC-8431, 1/250), mouse monoclonal anti human Keratin 14 (clone LL002, Novocastra, Catalog no. NCL-LL002, 1/200). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Goat anti-mouse Alexa Fluor 488 (Invitrogen, Catalog no. A-11001) was used as a secondary antibody. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Following PBS washes, coverslips were dried and mounted on a microscope slide using ProLong Diamond antifade mounting medium containing DAPI (ThermoFisher Scientific, Catalog no. P36962) and cured for 24 h at room temperature in the dark. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Images were collected with Zeiss 880 or 980 confocal microscopes using the ZEN software (Zeiss). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Confocal images were processed using Fiji software. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Genome engineering for each SNP (rs11119348, rs661849, and rs10984103) in iPSCs or GMSM-K was achieved using a previously described protocol. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Sequences of CRISPR RNAs (crRNA) and repair templates specific to each SNPs are provided in (Supplementary Data 24). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Briefly, equimolar concentrations of specific crRNA and trans-activating crRNA (tracrRNA, IDT, Catalog no. 1072532) were annealed at 95 °C for 5 min, followed by cooling at room temperature (RT) to form functional gRNA duplexes. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Further, the ribonucleoprotein (RNP) complex was prepared 15 min before transfection by mixing gRNAs (1.5 µM) with Cas9 protein (0.45 µM) (Sigma, Catalog no. CAS9PROT for iPSCs and IDT, Catalog no. 1081058 for GMSM-K) at RT. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | RNP complexes together with the repair template were transfected into 1 million iPSCs with Amaxa nucleofector (Human Stem Cell kit 2, Lonza, Catalog no. VPH-5022, program: A-023 for iPSCs) in the presence of ROCK inhibitor (Y-27632 – 10 mM stock, Stemgent, Catalog no. 04-0012-10). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Lonza Kit V, program: T-020 was used for transfecting GMSM-K. Individual colonies were hand-picked and plated into 96-well plates. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | DNA was extracted using Quick Extract DNA extraction solution (Epicentre, Catalog no. QE09050). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Colonies were screened by PCR and sequenced using primers flanking each SNP (Primer sequences are provided in Supplementary Data 25). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | From iPSCs transfection experiment, two independent clones each of homozygous risk (AA for rs11119348; CC for rs661849) and homozygous non-risk (CC for rs11119348; TT for rs661849) for each SNP near IRF6, and three independent clones of each of homozygous risk (CC) and non-risk (AA) genotype for rs10984103 near FOXE1 were isolated (Supplementary Figs. 13a, b, 16a). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | From the GMSM-K transfection experiment, multiple independent clones, seven clones of homozygous risk (AA for rs11119348), six clones of homozygous non-risk (CC for rs11119348) and four independent clones each of the homozygous risk (CC for rs661849), and homozygous non-risk (TT for rs661849) were isolated. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Deletion of FOXE1 gene in GMSM-K was achieved using a pair of gRNA (Supplementary Fig. 19). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | A set of three primers was used for colony screening, and three independent clones were isolated (Supplementary Data 26). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Sequencing of three independent clones revealed that all but 30 bp at the 3’ end of the single coding exon gene was deleted (Supplementary Fig. 19). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Two independent samples of each genotype (two clones of the homozygous risk genotype, and two clones of the homozygous non-risk genotype) for both SNPs at IRF6 locus and three independent samples of each genotype (three clones of the homozygous risk genotype, and three clones of the homozygous non-risk genotype) for rs10984103 at FOXE1 locus were seeded in triplicate in 12-well plates (50,000 cells per well). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Additionally, three independent samples of heterozygous genotype for each SNP individually were seeded (Supplementary Figs. 13a, b, 16a). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Further, the cells were induced to differentiate into embryonic oral epithelial (iOE) cells using a 10-day differentiation protocol described previously (Supplementary Figs. 13a, b, 16a). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Briefly, on the first day of differentiation stem cell media is replaced with media consisted of EpiCult-C media (StemCell Technologies, Catalog no. 05630) mixed with EpiLife (Thermo, Catalog no. MEPI500CA) at 1:1 ratio, supplemented with 0.1x supplement S7 (Thermo, Catalog no. S0175), 0.1 μM β-mercaptoethanol (Sigma, Catalog no. M7522) and 400 μM Smoothened agonist (Selleckchem, Catalog no. S7779). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | 150pM of Bone Morphogenic Protein-4 (R&D Systems, Catalog no. 314-BP-010) is continuously added daily starting from day 3 to day 7 of differentiation. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | At day 8, the media is supplemented with 1 uM of BMP-I inhibitor (LDN-193189) (Tocris, Catalog no. 6053), 5 uM of GSK3-Inhibitor (CHIR99021) (Selleckchem, Catalog no. 4423), 500 pM Epidermal Growth Factor (R&D systems, Catalog no. 236-EG) and 3.5 μM of Neurotrophin-4 (R&D systems, Catalog no. 268-N4). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The cultures were then harvested at day 10 at an oral epithelium stage. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | RNA was extracted from multiple samples of each genotype (homozygous risk and homozygous non-risk) for rs11119348, rs661849 and rs10984103 (iOE cells—six each genotype for both SNPs near IRF6 and nine each genotype for rs10984103 near FOXE1; GMSM-K—six non-risk and seven risk genotype for rs11119348 and four each genotype for rs661849) using Quick-DNA/RNA Miniprep kit (Zymo Research, Catalog no. D7001) followed by Dnase treatment using manufacturer’s instructions (Thermo Fisher Scientific, Catalog no. AM1907). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Reverse transcription was performed using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Catalog no. 4368814). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Real-time PCR was performed using SYBR Green qPCR mix (Bio-Rad) in the Bio-Rad CFX Connect Real-Time System. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Quantitative RT-PCR reaction for each sample was performed in triplicate. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Data analysis was performed using previously described methods. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Briefly, expression levels of IRF6 were normalized against ACTB for GMSM-K and expression levels of IRF6 or FOXE1 were normalized against ACTB, GAPDH, HPRT1, UBC and CDH1 for iOE cells (mentioned in the respective figure legends). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Data were presented as mean ± SD. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Statistical significance was determined using a two-tailed Student’s t-test. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Primer sequences used in the study for qRT-PCR are provided in Supplementary Data 27. |
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