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Article ; Online: Massively Parallel Profiling of Accessible Chromatin and Proteins with ASAP-Seq.

Mimitou, Eleni P / Smibert, Peter / Lareau, Caleb A

Methods in molecular biology (Clifton, N.J.)

2023 Volume 2611, Page(s) 249–267

Abstract: While methods such as the Assay for Transposase Accessible Chromatin by sequencing (ATAC-seq) enable a comprehensive characterization of regulatory DNA, additional measurements are required to characterize the multifaceted nature of eukaryotic cells. ... ...

Abstract	While methods such as the Assay for Transposase Accessible Chromatin by sequencing (ATAC-seq) enable a comprehensive characterization of regulatory DNA, additional measurements are required to characterize the multifaceted nature of eukaryotic cells. Here, we delineate the ATAC with Select Antigen Profiling by sequencing (ASAP-seq) protocol, a scalable approach to quantifying proteins via oligo-tagged antibodies alongside accessible DNA in thousands of single cells. Critically, our method utilizes a custom bridge oligo that enables the utilization of a variety of oligo-conjugated antibodies, enabling the utilization and repurposing of other commercial products. The ASAP-seq method can be completed with straightforward experimental and computational modifications existing single-cell ATAC-seq workflows but yields distinct modalities underlying complex cellular states, including estimation of protein abundance on the cell surface as well as intracellular and intranuclear factors.
MeSH term(s)	Chromatin ; High-Throughput Nucleotide Sequencing/methods ; Sequence Analysis, DNA/methods ; DNA/genetics ; Chromatin Immunoprecipitation Sequencing
Chemical Substances	Chromatin ; DNA (9007-49-2)
Language	English
Publishing date	2023-02-15
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-2899-7_13
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Direct detection of RNA modifications and structure using single-molecule nanopore sequencing.

Stephenson, William / Razaghi, Roham / Busan, Steven / Weeks, Kevin M / Timp, Winston / Smibert, Peter

Cell genomics

2022 Volume 2, Issue 2

Abstract: Modifications are present on many classes of RNA, including tRNA, rRNA, and mRNA. These modifications modulate diverse biological processes such as genetic recoding and mRNA export and folding. In addition, modifications can be introduced to RNA ... ...

Abstract	Modifications are present on many classes of RNA, including tRNA, rRNA, and mRNA. These modifications modulate diverse biological processes such as genetic recoding and mRNA export and folding. In addition, modifications can be introduced to RNA molecules using chemical probing strategies that reveal RNA structure and dynamics. Many methods exist to detect RNA modifications by short-read sequencing; however, limitations on read length inherent to short-read-based methods dissociate modifications from their native context, preventing single-molecule modification analysis. Here, we demonstrate direct RNA nanopore sequencing to detect endogenous and exogenous RNA modifications on long RNAs at the single-molecule level. We detect endogenous 2'-
Language	English
Publishing date	2022-02-07
Publishing country	United States
Document type	Journal Article
ISSN	2666-979X
ISSN (online)	2666-979X
DOI	10.1016/j.xgen.2022.100097
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Article ; Online: An open source toolkit for repurposing Illumina sequencing systems as versatile fluidics and imaging platforms.

Pandit, Kunal / Petrescu, Joana / Cuevas, Miguel / Stephenson, William / Smibert, Peter / Phatnani, Hemali / Maniatis, Silas

Scientific reports

2022 Volume 12, Issue 1, Page(s) 5081

Abstract: Fluorescence microscopy is a key method in the life sciences. State of the art -omics methods combine fluorescence microscopy with complex protocols to visualize tens to thousands of features in each of millions of pixels across samples. These -omics ... ...

Abstract	Fluorescence microscopy is a key method in the life sciences. State of the art -omics methods combine fluorescence microscopy with complex protocols to visualize tens to thousands of features in each of millions of pixels across samples. These -omics methods require precise control of temperature, reagent application, and image acquisition parameters during iterative chemistry and imaging cycles conducted over the course of days or weeks. Automated execution of such methods enables robust and reproducible data generation. However, few commercial solutions exist for temperature controlled, fluidics coupled fluorescence imaging, and implementation of bespoke instrumentation requires specialized engineering expertise. Here we present PySeq2500, an open source Python code base and flow cell design that converts the Illumina HiSeq 2500 instrument, comprising an epifluorescence microscope with integrated fluidics, into an open platform for programmable applications without need for specialized engineering or software development expertise. Customizable PySeq2500 protocols enable experimental designs involving simultaneous 4-channel image acquisition, temperature control, reagent exchange, stable positioning, and sample integrity over extended experiments. To demonstrate accessible automation of complex, multi-day workflows, we use the PySeq2500 system for unattended execution of iterative indirect immunofluorescence imaging (4i). Our automated 4i method uses off-the-shelf antibodies over multiple cycles of staining, imaging, and antibody elution to build highly multiplexed maps of cell types and pathological features in mouse and postmortem human spinal cord sections. Given the widespread availability of HiSeq 2500 platforms and the simplicity of the modifications required to repurpose these systems, PySeq2500 enables non-specialists to develop and implement state of the art fluidics coupled imaging methods in a widely available benchtop system.
MeSH term(s)	Animals ; Feces ; High-Throughput Nucleotide Sequencing/methods ; Mice ; Optical Imaging ; Software ; Workflow
Language	English
Publishing date	2022-03-24
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-022-08740-w
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Article ; Online: An open source toolkit for repurposing Illumina sequencing systems as versatile fluidics and imaging platforms

Kunal Pandit / Joana Petrescu / Miguel Cuevas / William Stephenson / Peter Smibert / Hemali Phatnani / Silas Maniatis

Scientific Reports, Vol 12, Iss 1, Pp 1-

2022 Volume 13

Abstract: Abstract Fluorescence microscopy is a key method in the life sciences. State of the art -omics methods combine fluorescence microscopy with complex protocols to visualize tens to thousands of features in each of millions of pixels across samples. These - ... ...

Abstract	Abstract Fluorescence microscopy is a key method in the life sciences. State of the art -omics methods combine fluorescence microscopy with complex protocols to visualize tens to thousands of features in each of millions of pixels across samples. These -omics methods require precise control of temperature, reagent application, and image acquisition parameters during iterative chemistry and imaging cycles conducted over the course of days or weeks. Automated execution of such methods enables robust and reproducible data generation. However, few commercial solutions exist for temperature controlled, fluidics coupled fluorescence imaging, and implementation of bespoke instrumentation requires specialized engineering expertise. Here we present PySeq2500, an open source Python code base and flow cell design that converts the Illumina HiSeq 2500 instrument, comprising an epifluorescence microscope with integrated fluidics, into an open platform for programmable applications without need for specialized engineering or software development expertise. Customizable PySeq2500 protocols enable experimental designs involving simultaneous 4-channel image acquisition, temperature control, reagent exchange, stable positioning, and sample integrity over extended experiments. To demonstrate accessible automation of complex, multi-day workflows, we use the PySeq2500 system for unattended execution of iterative indirect immunofluorescence imaging (4i). Our automated 4i method uses off-the-shelf antibodies over multiple cycles of staining, imaging, and antibody elution to build highly multiplexed maps of cell types and pathological features in mouse and postmortem human spinal cord sections. Given the widespread availability of HiSeq 2500 platforms and the simplicity of the modifications required to repurpose these systems, PySeq2500 enables non-specialists to develop and implement state of the art fluidics coupled imaging methods in a widely available benchtop system.
Keywords	Medicine ; R ; Science ; Q
Subject code	004
Language	English
Publishing date	2022-03-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Direct detection of RNA modifications and structure using single-molecule nanopore sequencing

William Stephenson / Roham Razaghi / Steven Busan / Kevin M. Weeks / Winston Timp / Peter Smibert

Cell Genomics, Vol 2, Iss 2, Pp 100097- (2022)

2022

Abstract: Summary: Modifications are present on many classes of RNA, including tRNA, rRNA, and mRNA. These modifications modulate diverse biological processes such as genetic recoding and mRNA export and folding. In addition, modifications can be introduced to RNA ...

Abstract	Summary: Modifications are present on many classes of RNA, including tRNA, rRNA, and mRNA. These modifications modulate diverse biological processes such as genetic recoding and mRNA export and folding. In addition, modifications can be introduced to RNA molecules using chemical probing strategies that reveal RNA structure and dynamics. Many methods exist to detect RNA modifications by short-read sequencing; however, limitations on read length inherent to short-read-based methods dissociate modifications from their native context, preventing single-molecule modification analysis. Here, we demonstrate direct RNA nanopore sequencing to detect endogenous and exogenous RNA modifications on long RNAs at the single-molecule level. We detect endogenous 2′-O-methyl and base modifications across E. coli and S. cerevisiae ribosomal RNAs as shifts in current signal and dwell times distally through interactions with the helicase motor protein. We further use the 2′-hydroxyl reactive SHAPE reagent acetylimidazole to probe RNA structure at the single-molecule level with readout by direct nanopore sequencing.
Keywords	RNA ; nanopore ; direct RNA nanopore sequencing ; RNA modifications ; RNA structure ; long-read ; Genetics ; QH426-470 ; Internal medicine ; RC31-1245
Subject code	612
Language	English
Publishing date	2022-02-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Characterizing cellular heterogeneity in chromatin state with scCUT&Tag-pro.

Zhang, Bingjie / Srivastava, Avi / Mimitou, Eleni / Stuart, Tim / Raimondi, Ivan / Hao, Yuhan / Smibert, Peter / Satija, Rahul

Nature biotechnology

2022 Volume 40, Issue 8, Page(s) 1220–1230

Abstract: Technologies that profile chromatin modifications at single-cell resolution offer enormous promise for functional genomic characterization, but the sparsity of the measurements and integrating multiple binding maps represent substantial challenges. Here ... ...

Abstract	Technologies that profile chromatin modifications at single-cell resolution offer enormous promise for functional genomic characterization, but the sparsity of the measurements and integrating multiple binding maps represent substantial challenges. Here we introduce single-cell (sc)CUT&Tag-pro, a multimodal assay for profiling protein-DNA interactions coupled with the abundance of surface proteins in single cells. In addition, we introduce single-cell ChromHMM, which integrates data from multiple experiments to infer and annotate chromatin states based on combinatorial histone modification patterns. We apply these tools to perform an integrated analysis across nine different molecular modalities in circulating human immune cells. We demonstrate how these two approaches can characterize dynamic changes in the function of individual genomic elements across both discrete cell states and continuous developmental trajectories, nominate associated motifs and regulators that establish chromatin states and identify extensive and cell-type-specific regulatory priming. Finally, we demonstrate how our integrated reference can serve as a scaffold to map and improve the interpretation of additional scCUT&Tag datasets.
MeSH term(s)	Chromatin/genetics ; Chromatin Immunoprecipitation ; DNA ; Genomics ; Histones/genetics ; Histones/metabolism ; Humans
Chemical Substances	Chromatin ; Histones ; DNA (9007-49-2)
Language	English
Publishing date	2022-03-24
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	1311932-1
ISSN	1546-1696 ; 1087-0156
ISSN (online)	1546-1696
ISSN	1087-0156
DOI	10.1038/s41587-022-01250-0
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Article ; Online: Integrating single-cell transcriptomic data across different conditions, technologies, and species.

Butler, Andrew / Hoffman, Paul / Smibert, Peter / Papalexi, Efthymia / Satija, Rahul

Nature biotechnology

2018 Volume 36, Issue 5, Page(s) 411–420

Abstract: Computational single-cell RNA-seq (scRNA-seq) methods have been successfully applied to experiments representing a single condition, technology, or species to discover and define cellular phenotypes. However, identifying subpopulations of cells that are ... ...

Abstract	Computational single-cell RNA-seq (scRNA-seq) methods have been successfully applied to experiments representing a single condition, technology, or species to discover and define cellular phenotypes. However, identifying subpopulations of cells that are present across multiple data sets remains challenging. Here, we introduce an analytical strategy for integrating scRNA-seq data sets based on common sources of variation, enabling the identification of shared populations across data sets and downstream comparative analysis. We apply this approach, implemented in our R toolkit Seurat (http://satijalab.org/seurat/), to align scRNA-seq data sets of peripheral blood mononuclear cells under resting and stimulated conditions, hematopoietic progenitors sequenced using two profiling technologies, and pancreatic cell 'atlases' generated from human and mouse islets. In each case, we learn distinct or transitional cell states jointly across data sets, while boosting statistical power through integrated analysis. Our approach facilitates general comparisons of scRNA-seq data sets, potentially deepening our understanding of how distinct cell states respond to perturbation, disease, and evolution.
MeSH term(s)	Animals ; Computers, Molecular ; Data Analysis ; Gene Expression Profiling ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Leukocytes, Mononuclear/chemistry ; Mice ; Sequence Analysis, RNA/methods ; Single-Cell Analysis/methods ; Software ; Transcriptome/genetics
Language	English
Publishing date	2018-04-02
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	1311932-1
ISSN	1546-1696 ; 1087-0156
ISSN (online)	1546-1696
ISSN	1087-0156
DOI	10.1038/nbt.4096
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Correction: Neural specificity of the RNA-binding protein Elav is achieved by post-transcriptional repression in non-neural tissues.

Sanfilippo, Piero / Smibert, Peter / Duan, Hong / Lai, Eric C

Development (Cambridge, England)

2017 Volume 144, Issue 8, Page(s) 1578

Language	English
Publishing date	2017--15
Publishing country	England
Document type	Journal Article ; Published Erratum
ZDB-ID	90607-4
ISSN	1477-9129 ; 0950-1991
ISSN (online)	1477-9129
ISSN	0950-1991
DOI	10.1242/dev.151753
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Article ; Online: Discovery of target genes and pathways at GWAS loci by pooled single-cell CRISPR screens.

Morris, John A / Caragine, Christina / Daniloski, Zharko / Domingo, Júlia / Barry, Timothy / Lu, Lu / Davis, Kyrie / Ziosi, Marcello / Glinos, Dafni A / Hao, Stephanie / Mimitou, Eleni P / Smibert, Peter / Roeder, Kathryn / Katsevich, Eugene / Lappalainen, Tuuli / Sanjana, Neville E

Science (New York, N.Y.)

2023 Volume 380, Issue 6646, Page(s) eadh7699

Abstract: Most variants associated with complex traits and diseases identified by genome-wide association studies (GWAS) map to noncoding regions of the genome with unknown effects. Using ancestrally diverse, biobank-scale GWAS data, massively parallel CRISPR ... ...

Abstract	Most variants associated with complex traits and diseases identified by genome-wide association studies (GWAS) map to noncoding regions of the genome with unknown effects. Using ancestrally diverse, biobank-scale GWAS data, massively parallel CRISPR screens, and single-cell transcriptomic and proteomic sequencing, we discovered 124
MeSH term(s)	Humans ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Predisposition to Disease ; Genome-Wide Association Study ; Polymorphism, Single Nucleotide ; Proteomics ; Quantitative Trait Loci ; Single-Cell Analysis ; Multifactorial Inheritance ; Blood Cells ; RNA-Seq ; Disease/genetics
Language	English
Publishing date	2023-05-19
Publishing country	United States
Document type	Journal Article
ZDB-ID	128410-1
ISSN	1095-9203 ; 0036-8075
ISSN (online)	1095-9203
ISSN	0036-8075
DOI	10.1126/science.adh7699
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Article ; Online: A genome-scale screen for synthetic drivers of T cell proliferation.

Legut, Mateusz / Gajic, Zoran / Guarino, Maria / Daniloski, Zharko / Rahman, Jahan A / Xue, Xinhe / Lu, Congyi / Lu, Lu / Mimitou, Eleni P / Hao, Stephanie / Davoli, Teresa / Diefenbach, Catherine / Smibert, Peter / Sanjana, Neville E

Nature

2022 Volume 603, Issue 7902, Page(s) 728–735

Abstract: The engineering of autologous patient T cells for adoptive cell therapies has revolutionized the treatment of several types of ... ...

Abstract	The engineering of autologous patient T cells for adoptive cell therapies has revolutionized the treatment of several types of cancer
MeSH term(s)	CD4-Positive T-Lymphocytes ; CD8-Positive T-Lymphocytes ; Cell Proliferation ; Humans ; Immunotherapy, Adoptive ; Lymphocyte Activation/genetics ; Neoplasms
Language	English
Publishing date	2022-03-16
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	120714-3
ISSN	1476-4687 ; 0028-0836
ISSN (online)	1476-4687
ISSN	0028-0836
DOI	10.1038/s41586-022-04494-7
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