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feature barcode technology  (10X Genomics)

 
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    Structured Review

    10X Genomics feature barcode technology
    Feature Barcode Technology, supplied by 10X Genomics, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/barcode/cellranger/pm42320984-60-38-41
    Average 86 stars, based on 1 article reviews
    feature barcode technology - by Bioz Stars, 2026-10
    86/100 stars

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    Related Articles

    Control:

    Article Title: Single-cell transcriptomics defines Dot1L interacting partners and downstream target genes in the mouse molar dental pulp.
    Article Snippet: .. Quality control for the scRna-seq FasTQ reads included: (1) filtering FASTQ reads with more than 1 bp mismatched barcode (10X Genomics, 2018) (2) filtering FASTQ reads with less than a 3 Q30 score (3) filtering STAR alignments with less than a 255 MAPQ score (dobin et al., 2013) samples were processed independently and then batch corrected via the BBKNN method (Polański et al., 2020). single-cell clustering procedures do not perfectly partition cell types, but instead indicate similarity of expression patterns which can be useful for exploring specific gene profiles of genes such as Dot1L. ..

    Single Cell:

    Article Title: Single-cell transcriptomics defines Dot1L interacting partners and downstream target genes in the mouse molar dental pulp.
    Article Snippet: .. Quality control for the scRna-seq FasTQ reads included: (1) filtering FASTQ reads with more than 1 bp mismatched barcode (10X Genomics, 2018) (2) filtering FASTQ reads with less than a 3 Q30 score (3) filtering STAR alignments with less than a 255 MAPQ score (dobin et al., 2013) samples were processed independently and then batch corrected via the BBKNN method (Polański et al., 2020). single-cell clustering procedures do not perfectly partition cell types, but instead indicate similarity of expression patterns which can be useful for exploring specific gene profiles of genes such as Dot1L. ..

    Expressing:

    Article Title: Single-cell transcriptomics defines Dot1L interacting partners and downstream target genes in the mouse molar dental pulp.
    Article Snippet: .. Quality control for the scRna-seq FasTQ reads included: (1) filtering FASTQ reads with more than 1 bp mismatched barcode (10X Genomics, 2018) (2) filtering FASTQ reads with less than a 3 Q30 score (3) filtering STAR alignments with less than a 255 MAPQ score (dobin et al., 2013) samples were processed independently and then batch corrected via the BBKNN method (Polański et al., 2020). single-cell clustering procedures do not perfectly partition cell types, but instead indicate similarity of expression patterns which can be useful for exploring specific gene profiles of genes such as Dot1L. ..

    other:

    Article Title: Substrate stiffness enhances human regulatory T cell induction and metabolism.
    Article Snippet: Regulatory T cells (Tregs) provide an essential tolerance mechanism to suppress the immune response.. Induced Tregs hold the potential to treat autoimmune diseases in adoptive therapy and can be produced with stimulating signals to CD3 and CD28 in presence of the cytokine TGF-β and IL-2.. This report examines the modulation of human Treg induction by leveraging the ability of T cells to sense the mechanical stiffness of an activating substrate.

    Article Title: Single-cell investigation of lead toxicity from neurodevelopment to neurodegeneration: Current review and future opportunities
    Article Snippet: Typically, each RNA transcript is barcoded with a unique molecular identifier that is associated with each individual droplet, assumed to be one single cell (e.g., barcode for 10x Genomics) based on dilution calculations.

    Isolation:

    Article Title: Neonatal immunity associated with heterologous HIV-1 neutralizing antibody induction in SHIV-infected Rhesus Macaques
    Article Snippet: .. We used an antigen-specific high-throughput platform referred to as barcode-enable antigen mapping for B cells (BEAM-Ab) from 10X Genomics (Pleasanton, CA) in our pipeline for isolation and characterization of HIV-1 Env-reactive mAbs (Fig. ). ..

    Amplification:

    Article Title: Single cell susceptibility to SARS-CoV-2 infection is driven by variable cell states
    Article Snippet: .. We extracted barcode information directly from the amplified cDNA from 10X Genomics V3 chemistry protocol (step 2). ..

    Incubation:

    Article Title: Protracted neuronal recruitment in the temporal lobes of young children
    Article Snippet: .. Each one of the five nuclei suspensions was incubated with its own unique CellPlex Cell Multiplexing Oligo (CMO) barcode (10x Genomics) for 5 min followed by a 500 g centrifugation at 4 °C for 10 min. After centrifugation, the supernatant was discarded and nuclei were gently resuspended in WRB using a P200 pipette for a minimum of 2 min, until a clean single-nuclei suspension was obtained. ..

    Multiplexing:

    Article Title: Protracted neuronal recruitment in the temporal lobes of young children
    Article Snippet: .. Each one of the five nuclei suspensions was incubated with its own unique CellPlex Cell Multiplexing Oligo (CMO) barcode (10x Genomics) for 5 min followed by a 500 g centrifugation at 4 °C for 10 min. After centrifugation, the supernatant was discarded and nuclei were gently resuspended in WRB using a P200 pipette for a minimum of 2 min, until a clean single-nuclei suspension was obtained. ..

    Centrifugation:

    Article Title: Protracted neuronal recruitment in the temporal lobes of young children
    Article Snippet: .. Each one of the five nuclei suspensions was incubated with its own unique CellPlex Cell Multiplexing Oligo (CMO) barcode (10x Genomics) for 5 min followed by a 500 g centrifugation at 4 °C for 10 min. After centrifugation, the supernatant was discarded and nuclei were gently resuspended in WRB using a P200 pipette for a minimum of 2 min, until a clean single-nuclei suspension was obtained. ..

    Transferring:

    Article Title: Protracted neuronal recruitment in the temporal lobes of young children
    Article Snippet: .. Each one of the five nuclei suspensions was incubated with its own unique CellPlex Cell Multiplexing Oligo (CMO) barcode (10x Genomics) for 5 min followed by a 500 g centrifugation at 4 °C for 10 min. After centrifugation, the supernatant was discarded and nuclei were gently resuspended in WRB using a P200 pipette for a minimum of 2 min, until a clean single-nuclei suspension was obtained. ..

    Suspension:

    Article Title: Protracted neuronal recruitment in the temporal lobes of young children
    Article Snippet: .. Each one of the five nuclei suspensions was incubated with its own unique CellPlex Cell Multiplexing Oligo (CMO) barcode (10x Genomics) for 5 min followed by a 500 g centrifugation at 4 °C for 10 min. After centrifugation, the supernatant was discarded and nuclei were gently resuspended in WRB using a P200 pipette for a minimum of 2 min, until a clean single-nuclei suspension was obtained. ..

    Cell Characterization:

    Article Title: Effects of Leydig cell elimination on testicular interstitial cell populations: characterization by scRNA-seq and immunocytochemical techniques
    Article Snippet: Cell capture, 10x Genomics library preparation, and sequencing were performed by Novogene (Beijing, China). .. After washing twice in PBS, approximately 10,000 cells were loaded onto 10x Chromium chips with 3′v2 chemistry and barcode to achieve a targeted cell count of 8,000, according to the manufacturer’s instructions (10x Genomics, Pleasanton, CA). ..



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    A. Schematic of the <t>Swap-seq</t> workflow for measuring the effects of DNA sequence deletions on gene expression. Each Swap-seq edit uses a pair of pegRNAs to delete a 50- to 250-bp target sequence and replace it with a <t>barcode</t> cassette containing an edit-specific 8-bp barcode together with sequences common to all edits, including PCR primer-binding sites (yellow) and a 3′ overlap sequence (purple). In a Swap-seq screen, hundreds of such edits are designed to perturb sequences surrounding a gene of interest. Cells are infected with a lentiviral pegRNA-pair library at low multiplicity of infection (MOI); after 14 days of editing, cells are cultured without doxycycline for 7 days before undergoing FlowFISH sorting and quantification of changes in gene expression. B. Individual Swap-seq editing test in three regions: the PPIF promoter, a candidate enhancer with high accessibility and H3K27ac signal (enhancer e2 in G ), and a region with low accessibility (hg19: chr10:81,069,000-81,069,517). Error bars: 95% CI for the mean. C. Correlation of effect sizes between two biological replicates, after removing edits that did not pass the 0.001% frequency threshold. Effects for each edit are averaged among barcodes. (Pearson’s r = 0.97, n = 117 edits). D. Editing efficiency and number of cells assayed for each barcode. A barcode passed the threshold in a given technical replicate if aligned reads exceeded 0.001% of all reads. Bottom axis, mean allele frequency calculated as the percent of reads aligned to the barcode averaged across replicates; y-axis, number of barcodes; top axis, number of cells assayed. E. Volcano plot of Swap-seq edits passing the frequency threshold. Edits were considered significant at BH-corrected p < 0.001 (horizontal dashed line). Error bars: 95% CI for the mean. F. Swap-seq screen candidate elements at the PPIF locus. In total, 24 candidate regulatory elements were targeted and the PPIF promoter was included as a positive control. *Two elements with no edits above the frequency threshold of 0.001%. G. Swap-seq effects on PPIF expression. Red, significant edits (BH-corrected p < 0.001, one-sample t-test); gray, non-significant edits. H. Swap-seq edits spanning the promoter, splice junction, and enhancer g1. Red, significant edits (BH-corrected p < 0.001, one-sample t-test); gray, non-significant edits.
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    A. Schematic of the <t>Swap-seq</t> workflow for measuring the effects of DNA sequence deletions on gene expression. Each Swap-seq edit uses a pair of pegRNAs to delete a 50- to 250-bp target sequence and replace it with a <t>barcode</t> cassette containing an edit-specific 8-bp barcode together with sequences common to all edits, including PCR primer-binding sites (yellow) and a 3′ overlap sequence (purple). In a Swap-seq screen, hundreds of such edits are designed to perturb sequences surrounding a gene of interest. Cells are infected with a lentiviral pegRNA-pair library at low multiplicity of infection (MOI); after 14 days of editing, cells are cultured without doxycycline for 7 days before undergoing FlowFISH sorting and quantification of changes in gene expression. B. Individual Swap-seq editing test in three regions: the PPIF promoter, a candidate enhancer with high accessibility and H3K27ac signal (enhancer e2 in G ), and a region with low accessibility (hg19: chr10:81,069,000-81,069,517). Error bars: 95% CI for the mean. C. Correlation of effect sizes between two biological replicates, after removing edits that did not pass the 0.001% frequency threshold. Effects for each edit are averaged among barcodes. (Pearson’s r = 0.97, n = 117 edits). D. Editing efficiency and number of cells assayed for each barcode. A barcode passed the threshold in a given technical replicate if aligned reads exceeded 0.001% of all reads. Bottom axis, mean allele frequency calculated as the percent of reads aligned to the barcode averaged across replicates; y-axis, number of barcodes; top axis, number of cells assayed. E. Volcano plot of Swap-seq edits passing the frequency threshold. Edits were considered significant at BH-corrected p < 0.001 (horizontal dashed line). Error bars: 95% CI for the mean. F. Swap-seq screen candidate elements at the PPIF locus. In total, 24 candidate regulatory elements were targeted and the PPIF promoter was included as a positive control. *Two elements with no edits above the frequency threshold of 0.001%. G. Swap-seq effects on PPIF expression. Red, significant edits (BH-corrected p < 0.001, one-sample t-test); gray, non-significant edits. H. Swap-seq edits spanning the promoter, splice junction, and enhancer g1. Red, significant edits (BH-corrected p < 0.001, one-sample t-test); gray, non-significant edits.
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    86
    10X Genomics feature barcode kit
    A. Schematic of the <t>Swap-seq</t> workflow for measuring the effects of DNA sequence deletions on gene expression. Each Swap-seq edit uses a pair of pegRNAs to delete a 50- to 250-bp target sequence and replace it with a <t>barcode</t> cassette containing an edit-specific 8-bp barcode together with sequences common to all edits, including PCR primer-binding sites (yellow) and a 3′ overlap sequence (purple). In a Swap-seq screen, hundreds of such edits are designed to perturb sequences surrounding a gene of interest. Cells are infected with a lentiviral pegRNA-pair library at low multiplicity of infection (MOI); after 14 days of editing, cells are cultured without doxycycline for 7 days before undergoing FlowFISH sorting and quantification of changes in gene expression. B. Individual Swap-seq editing test in three regions: the PPIF promoter, a candidate enhancer with high accessibility and H3K27ac signal (enhancer e2 in G ), and a region with low accessibility (hg19: chr10:81,069,000-81,069,517). Error bars: 95% CI for the mean. C. Correlation of effect sizes between two biological replicates, after removing edits that did not pass the 0.001% frequency threshold. Effects for each edit are averaged among barcodes. (Pearson’s r = 0.97, n = 117 edits). D. Editing efficiency and number of cells assayed for each barcode. A barcode passed the threshold in a given technical replicate if aligned reads exceeded 0.001% of all reads. Bottom axis, mean allele frequency calculated as the percent of reads aligned to the barcode averaged across replicates; y-axis, number of barcodes; top axis, number of cells assayed. E. Volcano plot of Swap-seq edits passing the frequency threshold. Edits were considered significant at BH-corrected p < 0.001 (horizontal dashed line). Error bars: 95% CI for the mean. F. Swap-seq screen candidate elements at the PPIF locus. In total, 24 candidate regulatory elements were targeted and the PPIF promoter was included as a positive control. *Two elements with no edits above the frequency threshold of 0.001%. G. Swap-seq effects on PPIF expression. Red, significant edits (BH-corrected p < 0.001, one-sample t-test); gray, non-significant edits. H. Swap-seq edits spanning the promoter, splice junction, and enhancer g1. Red, significant edits (BH-corrected p < 0.001, one-sample t-test); gray, non-significant edits.
    Feature Barcode Kit, supplied by 10X Genomics, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/barcode/3%CA%B9+barcode+feature+kit/pm42321208-228-27-34
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    barcode r  (Sangon Biotech)
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    A. Schematic of the <t>Swap-seq</t> workflow for measuring the effects of DNA sequence deletions on gene expression. Each Swap-seq edit uses a pair of pegRNAs to delete a 50- to 250-bp target sequence and replace it with a <t>barcode</t> cassette containing an edit-specific 8-bp barcode together with sequences common to all edits, including PCR primer-binding sites (yellow) and a 3′ overlap sequence (purple). In a Swap-seq screen, hundreds of such edits are designed to perturb sequences surrounding a gene of interest. Cells are infected with a lentiviral pegRNA-pair library at low multiplicity of infection (MOI); after 14 days of editing, cells are cultured without doxycycline for 7 days before undergoing FlowFISH sorting and quantification of changes in gene expression. B. Individual Swap-seq editing test in three regions: the PPIF promoter, a candidate enhancer with high accessibility and H3K27ac signal (enhancer e2 in G ), and a region with low accessibility (hg19: chr10:81,069,000-81,069,517). Error bars: 95% CI for the mean. C. Correlation of effect sizes between two biological replicates, after removing edits that did not pass the 0.001% frequency threshold. Effects for each edit are averaged among barcodes. (Pearson’s r = 0.97, n = 117 edits). D. Editing efficiency and number of cells assayed for each barcode. A barcode passed the threshold in a given technical replicate if aligned reads exceeded 0.001% of all reads. Bottom axis, mean allele frequency calculated as the percent of reads aligned to the barcode averaged across replicates; y-axis, number of barcodes; top axis, number of cells assayed. E. Volcano plot of Swap-seq edits passing the frequency threshold. Edits were considered significant at BH-corrected p < 0.001 (horizontal dashed line). Error bars: 95% CI for the mean. F. Swap-seq screen candidate elements at the PPIF locus. In total, 24 candidate regulatory elements were targeted and the PPIF promoter was included as a positive control. *Two elements with no edits above the frequency threshold of 0.001%. G. Swap-seq effects on PPIF expression. Red, significant edits (BH-corrected p < 0.001, one-sample t-test); gray, non-significant edits. H. Swap-seq edits spanning the promoter, splice junction, and enhancer g1. Red, significant edits (BH-corrected p < 0.001, one-sample t-test); gray, non-significant edits.
    Barcode R, supplied by Sangon Biotech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/barcode/barcode+f+linker/pmc13142104-76-0-5
    Average 86 stars, based on 1 article reviews
    barcode r - by Bioz Stars, 2026-10
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    A. Schematic of the Swap-seq workflow for measuring the effects of DNA sequence deletions on gene expression. Each Swap-seq edit uses a pair of pegRNAs to delete a 50- to 250-bp target sequence and replace it with a barcode cassette containing an edit-specific 8-bp barcode together with sequences common to all edits, including PCR primer-binding sites (yellow) and a 3′ overlap sequence (purple). In a Swap-seq screen, hundreds of such edits are designed to perturb sequences surrounding a gene of interest. Cells are infected with a lentiviral pegRNA-pair library at low multiplicity of infection (MOI); after 14 days of editing, cells are cultured without doxycycline for 7 days before undergoing FlowFISH sorting and quantification of changes in gene expression. B. Individual Swap-seq editing test in three regions: the PPIF promoter, a candidate enhancer with high accessibility and H3K27ac signal (enhancer e2 in G ), and a region with low accessibility (hg19: chr10:81,069,000-81,069,517). Error bars: 95% CI for the mean. C. Correlation of effect sizes between two biological replicates, after removing edits that did not pass the 0.001% frequency threshold. Effects for each edit are averaged among barcodes. (Pearson’s r = 0.97, n = 117 edits). D. Editing efficiency and number of cells assayed for each barcode. A barcode passed the threshold in a given technical replicate if aligned reads exceeded 0.001% of all reads. Bottom axis, mean allele frequency calculated as the percent of reads aligned to the barcode averaged across replicates; y-axis, number of barcodes; top axis, number of cells assayed. E. Volcano plot of Swap-seq edits passing the frequency threshold. Edits were considered significant at BH-corrected p < 0.001 (horizontal dashed line). Error bars: 95% CI for the mean. F. Swap-seq screen candidate elements at the PPIF locus. In total, 24 candidate regulatory elements were targeted and the PPIF promoter was included as a positive control. *Two elements with no edits above the frequency threshold of 0.001%. G. Swap-seq effects on PPIF expression. Red, significant edits (BH-corrected p < 0.001, one-sample t-test); gray, non-significant edits. H. Swap-seq edits spanning the promoter, splice junction, and enhancer g1. Red, significant edits (BH-corrected p < 0.001, one-sample t-test); gray, non-significant edits.

    Journal: bioRxiv

    Article Title: Systematic mapping of enhancers, silencers, and topological elements with prime editing deletion screens

    doi: 10.64898/2026.10.08.756856

    Figure Lengend Snippet: A. Schematic of the Swap-seq workflow for measuring the effects of DNA sequence deletions on gene expression. Each Swap-seq edit uses a pair of pegRNAs to delete a 50- to 250-bp target sequence and replace it with a barcode cassette containing an edit-specific 8-bp barcode together with sequences common to all edits, including PCR primer-binding sites (yellow) and a 3′ overlap sequence (purple). In a Swap-seq screen, hundreds of such edits are designed to perturb sequences surrounding a gene of interest. Cells are infected with a lentiviral pegRNA-pair library at low multiplicity of infection (MOI); after 14 days of editing, cells are cultured without doxycycline for 7 days before undergoing FlowFISH sorting and quantification of changes in gene expression. B. Individual Swap-seq editing test in three regions: the PPIF promoter, a candidate enhancer with high accessibility and H3K27ac signal (enhancer e2 in G ), and a region with low accessibility (hg19: chr10:81,069,000-81,069,517). Error bars: 95% CI for the mean. C. Correlation of effect sizes between two biological replicates, after removing edits that did not pass the 0.001% frequency threshold. Effects for each edit are averaged among barcodes. (Pearson’s r = 0.97, n = 117 edits). D. Editing efficiency and number of cells assayed for each barcode. A barcode passed the threshold in a given technical replicate if aligned reads exceeded 0.001% of all reads. Bottom axis, mean allele frequency calculated as the percent of reads aligned to the barcode averaged across replicates; y-axis, number of barcodes; top axis, number of cells assayed. E. Volcano plot of Swap-seq edits passing the frequency threshold. Edits were considered significant at BH-corrected p < 0.001 (horizontal dashed line). Error bars: 95% CI for the mean. F. Swap-seq screen candidate elements at the PPIF locus. In total, 24 candidate regulatory elements were targeted and the PPIF promoter was included as a positive control. *Two elements with no edits above the frequency threshold of 0.001%. G. Swap-seq effects on PPIF expression. Red, significant edits (BH-corrected p < 0.001, one-sample t-test); gray, non-significant edits. H. Swap-seq edits spanning the promoter, splice junction, and enhancer g1. Red, significant edits (BH-corrected p < 0.001, one-sample t-test); gray, non-significant edits.

    Article Snippet: Following extraction, we split genomic DNA into PCR replicates and amplified the Swap-seq barcode cassettes with Q5 High-Fidelity 2X Master Mix (New England Biolabs #M0492L) and 0.5 uM of each amplification primer ( table S1 ) with the following program: 98°C for 1 minute and then 26 cycles of 98°C for 10 seconds, 69°C for 20 seconds, 72°C for 20 seconds, followed by a final 72°C extension for 2 minutes.

    Techniques: Sequencing, Gene Expression, Binding Assay, Infection, Cell Culture, Positive Control, Expressing, Polymerase Chain Reaction, ChIP-sequencing

    A. Comparison of regulatory elements identified by Swap-seq and CRISPRi. An element was classified as significant in Swap-seq if at least one edit within the element was significant (one-sample t-test, BH-corrected p < 0.001). CRISPRi used the average effects of all gRNAs targeting the same region (one-sample t-test, BH-corrected p < 0.05). Elements were grouped according to the hypothesized mechanisms based on their candidate element categories and effects on PPIF gene expression. B. Correlation between Swap-seq and CRISPRi effect sizes. Elements within the PPIF gene body, which are not testable by CRISPRi, were excluded from this analysis; when an element contains several significant edits, the one with the strongest effect was used for comparison with CRISPRi (Pearson’s r = 0.75, n = 18 elements). Error bars: 95% CI for the mean. C. Swap-seq edits overlap key transcription factor binding sites (TFBS) within regulatory elements. Close-up views of four elements with significant effects on PPIF expression (left to right: e2, a PPIF enhancer with high H3K27ac; e5, a low-H3K27ac enhancer; g1, an enhancer in intron 1 of PPIF ; c2, a CTCF binding site). ChromBPNet attribution scores are plotted together with TF-MoDISco motif annotations. All edits that passed the frequency threshold are plotted with width corresponding to the deleted region. Significance determined by one-sample t-test: red, significant edits (BH-corrected p < 0.001); gray, non-significant edits. Error bars: 95% CI for the mean. D. Schematic of the downstream lentiMPRA. Candidate regulatory elements were cloned downstream of the promoter and an ORF–barcode–poly(A) cassette (ORF-BC-pA). Cells were infected with the lentiviral library at high MOI, and barcodes were sequenced from both DNA and RNA; CRE activity was calculated as the ratio of RNA to DNA barcode abundance. E. LentiMPRA validation of candidate enhancers. Each dot represents a barcode from two biological replicates. A sequence lacking predicted TFBS (noTFBS) served as a negative control for basal promoter strength. Significance determined by two-sample t-test: *p < 0.05, **p < 0.01, ***p < 0.001. Error bars: 95% CI for the mean. F. CTCF deletion alters 3D genome contacts at the PPIF locus. Contact frequencies were averaged across region-capture Micro-C (RCMC) measurements from three independent clones, and log2 fold-change was calculated as log2(c2-deleted clones / WT clones) at 1.6 kb bin sizes. G. Quantification of contact changes associated with CTCF deletion, with the viewpoint on the PPIF promoter. Bars show changes in contact frequency between the PPIF promoter and each regulatory element, averaged across three clonal lines at 1.6 kb binsize. Significance determined by two-sample t-test: *p < 0.05, **p < 0.01, ***p < 0.001. Error bars: 95% CI for the mean.

    Journal: bioRxiv

    Article Title: Systematic mapping of enhancers, silencers, and topological elements with prime editing deletion screens

    doi: 10.64898/2026.10.08.756856

    Figure Lengend Snippet: A. Comparison of regulatory elements identified by Swap-seq and CRISPRi. An element was classified as significant in Swap-seq if at least one edit within the element was significant (one-sample t-test, BH-corrected p < 0.001). CRISPRi used the average effects of all gRNAs targeting the same region (one-sample t-test, BH-corrected p < 0.05). Elements were grouped according to the hypothesized mechanisms based on their candidate element categories and effects on PPIF gene expression. B. Correlation between Swap-seq and CRISPRi effect sizes. Elements within the PPIF gene body, which are not testable by CRISPRi, were excluded from this analysis; when an element contains several significant edits, the one with the strongest effect was used for comparison with CRISPRi (Pearson’s r = 0.75, n = 18 elements). Error bars: 95% CI for the mean. C. Swap-seq edits overlap key transcription factor binding sites (TFBS) within regulatory elements. Close-up views of four elements with significant effects on PPIF expression (left to right: e2, a PPIF enhancer with high H3K27ac; e5, a low-H3K27ac enhancer; g1, an enhancer in intron 1 of PPIF ; c2, a CTCF binding site). ChromBPNet attribution scores are plotted together with TF-MoDISco motif annotations. All edits that passed the frequency threshold are plotted with width corresponding to the deleted region. Significance determined by one-sample t-test: red, significant edits (BH-corrected p < 0.001); gray, non-significant edits. Error bars: 95% CI for the mean. D. Schematic of the downstream lentiMPRA. Candidate regulatory elements were cloned downstream of the promoter and an ORF–barcode–poly(A) cassette (ORF-BC-pA). Cells were infected with the lentiviral library at high MOI, and barcodes were sequenced from both DNA and RNA; CRE activity was calculated as the ratio of RNA to DNA barcode abundance. E. LentiMPRA validation of candidate enhancers. Each dot represents a barcode from two biological replicates. A sequence lacking predicted TFBS (noTFBS) served as a negative control for basal promoter strength. Significance determined by two-sample t-test: *p < 0.05, **p < 0.01, ***p < 0.001. Error bars: 95% CI for the mean. F. CTCF deletion alters 3D genome contacts at the PPIF locus. Contact frequencies were averaged across region-capture Micro-C (RCMC) measurements from three independent clones, and log2 fold-change was calculated as log2(c2-deleted clones / WT clones) at 1.6 kb bin sizes. G. Quantification of contact changes associated with CTCF deletion, with the viewpoint on the PPIF promoter. Bars show changes in contact frequency between the PPIF promoter and each regulatory element, averaged across three clonal lines at 1.6 kb binsize. Significance determined by two-sample t-test: *p < 0.05, **p < 0.01, ***p < 0.001. Error bars: 95% CI for the mean.

    Article Snippet: Following extraction, we split genomic DNA into PCR replicates and amplified the Swap-seq barcode cassettes with Q5 High-Fidelity 2X Master Mix (New England Biolabs #M0492L) and 0.5 uM of each amplification primer ( table S1 ) with the following program: 98°C for 1 minute and then 26 cycles of 98°C for 10 seconds, 69°C for 20 seconds, 72°C for 20 seconds, followed by a final 72°C extension for 2 minutes.

    Techniques: Comparison, Gene Expression, Analysis, Binding Assay, Expressing, Clone Assay, Infection, Activity Assay, Biomarker Discovery, Sequencing, Negative Control