![]() (D) Percentage of DNA reads aligning to each chromosome from SPRITE clusters containing the Xist lncRNA (black) as compared to all SPRITE clusters (gray). Distributions shown for all clusters (left) and paired clusters (2+ reads per cluster) (right). (C) Percentage of reads in SPRITE clusters of different sizes, stratified into categories of clusters containing 1, 2–10, 11–100, 101–1000, and 1001+ reads per cluster. (B) The percentage of reads aligning to each DNA strand based on their DPM tag (DNA reads) or RPM tag (RNA reads) is shown across 144 independently amplified and sequenced SPRITE libraries from four SPRITE experiments (technical replicates). RPM and DPM tags have identical dsDNA sticky ends that enable subsequent split-pool barcoding with the same SPRITE tags. DNA is double stranded and therefore DPM will be read from both strands, while RNA is single stranded and therefore RPM will be read only from 1 strand. ![]() DNA and RNA are each tagged with sequence-specific tags, namely DPM tag and RPM tags using T4 DNA and RNA Ligase, respectively. (A) Schematic of tagging used to identify DNA- and RNA-specific reads through sequencing. The co-second authors agree to listing their names in the second position on CVs, resumes, and presentations.ġ: Supplemental Figure 1: RD-SPRITE accurately measures RNA and DNA contacts, Related to Figure 1. and oversaw all experiments and analysis performed computational analysis and generated scripts for analyzing the RD-SPRITE data and wrote the paper. provided guidance and support on imaging, analysis, ideas, and discussions on the paper. helped develop and maintain engineered cell lines used in this study aided in the preparation of samples used in this study. performed all live-cell 3D-SIM imaging and analysis of FL-SHARP and ΔRRM-SHARP localization. developed all engineered cell lines used in this study, including the doxycycline inducible Xist cell lines, Kcnq1ot1 lines, SHARP binding site deletions, and dCas9 cell lines. led the effort on the data processing and curation, writing scripts and constructing pipelines that enabled data interpretation of the RD-SPRITE dataset was responsible for gene, repeat, and allele annotation as well as validation and producing several QC metrics contributed to experimental optimization of the RNA-DNA SPRITE protocol. to perform SHARP purifications for Kcnq1ot1 binding advised and helped to develop and optimize the RNA molecular biology of the RD-SPRITE method in this project. performed the actinomycin D RD-SPRITE and DNA-SPRITE experiments performed analysis on the H3K27ac ChIP-seq developed the engineered SHARP lines for CLAP and methods for purification of SHARP worked with A.K.B. performed the data processing and analysis of the actinomycin D-treated SPRITE datasets developed statistical methods for multiway RNA analyses, created new methods for data analysis and visualization wrote scripts and constructed pipelines that enabled data curation wrote and provided comments and edits for the manuscript, figures, supplemental tables, and methods. to develop and characterize the inducible Kcnq1ot1 cell line and to generate homozygous deletions of the SHARP Binding Site within Kcnq1ot1 worked with MRB to purify SHARP and map it to Kcnq1ot1. performed all Kcnq1ot1 biochemical and functional experiments, including CRISPRi knockdowns, TSA treatments, H3K27ac ChIP-seq and functional characterizations worked with A.C. ![]() led the effort to analyze and interpret data, wrote software, created new methods for data analysis and visualization, performed analysis and visualization on the data and contributed major findings and results, created main and supplemental figures, and contributed to the initial draft of paper, model schematics/illustrations, and reviewed and edited the manuscript. developed and optimized the RD-SPRITE protocol, performed SPRITE experiments, analyzed and interpreted data, contributed to data visualization, figure presentation, model schematics/illustrations, and wrote the paper. designed, performed, acquired, and analyzed all the RNA-FISH, DNA-FISH, IF, IF/RNA-FISH experiments and made all imaging figures performed all LNA-related experiments and generated the figures and results performed actinomycin D and Flavopiridol treatments and analysis performed imaging of SHARP and Kcnq1ot1/Nap1l4 localization contributed to the writing of the centromeric RNA hub section, model schematics/illustrations, and provided comments and edits on the entire manuscript. conceived of this project with M.G., led the development and optimization of the RD-SPRITE method, performed experiments, analyzed and interpreted data, generated figures, oversaw all aspects of the project, and wrote the paper.
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