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SRP043036
Saccharomyces cerevisiae
12 Downloadable Samples
Illumina HiSeq 2000, Illumina Genome Analyzer II
Description
During translation elongation, the ribosome ratchets along its mRNA template, incorporating each new amino acid and translocating from one codon to the next. The elongation cycle requires dramatic structural rearrangements of the ribosome. We show here that deep sequencing of ribosome-protected mRNA fragments reveals not only the position of each ribosome but also, unexpectedly, its particular stage of the elongation cycle. Sequencing reveals two distinct populations of ribosome footprints, 28-30 nucleotides and 20-22 nucleotides long, representing translating ribosomes in distinct states, differentially stabilized by specific elongation inhibitors. We find that the balance of small and large footprints varies by codon and is correlated with translation speed. The ability to visualize conformational changes in the ribosome during elongation, at single-codon resolution, provides a new way to study the detailed kinetics of translation and a new probe with which to identify the factors that affect each step in the elongation cycle. Overall design: Ribosome profiling, or sequencing of ribosome-protected mRNA fragments, in yeast. We assay ribosome footprint sizes and positions in three conditions: untreated yeast (3 replicates) and yeast treated with translation inhibitors cycloheximide (2 replicates) and anisomycin (2 biological replicates, one technical replicate). We also treat yeast with 3-aminotriazole to measure the effect of limited histidine tRNAs on ribosome footprint size and distribution (two treatment durations).
Publication Title
Distinct stages of the translation elongation cycle revealed by sequencing ribosome-protected mRNA fragments.
Sample Metadata Fields
Cell line, Treatment, Subject
View Samples- Homo sapiens
- 26 Downloadable Samples
- Illumina HiSeq 2500
Description
Base Editing has been touted the most intelligent and precise application of the CRISPR platform so far, merging the simplicity of RNA-guided nucleases with deaminases that allow for the programmable generation of single base substitutions - without introduction of double-strand breaks. Even though the two-component system has been expected to cause off-target substitutions, studies involving cytosine base editors (CBEs) showed that in most cases, relatively few single base off-targets could be detected on DNA. We introduce the concept of multi-dimensional off-targeting, presenting an extensive amount of RNA cytidines being edited by DNA base editors. Epitranscriptomic off-target effects affected different cell lines and were independent of the guide RNAs used, suggesting Cas9-independent activity of the cytidine deaminase rAPOBEC1 on single-stranded RNA. With the help of protein engineering, we developed CBE variants with massively reduced inadvertent mutation of RNA that preserve and enhance DNA base editing capabilities. Overall design: HEK293T and HepG2 cells were transfected with regular and modified pCAG-BE3-P2A-EGFP or control pCAG-nCas9(D10A)-UGI-NLS-P2A-EGFP or control pCAG-P2A-EGFP constructs with various gRNAs as described below. Cells were sorted for top 5% GFP or all GFP + cells based on FITC signal. RNA-seq was performed to measure transcriptional changes associated with different constructs and guides.
Publication Title
Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors.
Sample Metadata Fields
Cell line, Treatment, Subject
View Samples- Homo sapiens
- 21 Downloadable Samples
- Illumina HiSeq 2500
Description
Base Editing has been touted the most intelligent and precise application of the CRISPR platform so far, merging the simplicity of RNA-guided nucleases with deaminases that allow for the programmable generation of single base substitutions - without introduction of double-strand breaks. Even though the two-component system has been expected to cause off-target substitutions, studies involving cytosine base editors (CBEs) showed that in most cases, relatively few single base off-targets could be detected on DNA. We introduce the concept of multi-dimensional off-targeting, presenting an extensive amount of RNA cytidines being edited by DNA base editors. Epitranscriptomic off-target effects affected different cell lines and were independent of the guide RNAs used, suggesting Cas9-independent activity of the cytidine deaminase rAPOBEC1 on single-stranded RNA. With the help of protein engineering, we developed CBE variants with massively reduced inadvertent mutation of RNA that preserve and enhance DNA base editing capabilities. Overall design: HEK293T and HepG2 cells were transfected with pCAG-BE3-P2A-EGFP or control pCAG-nCas9(D10A)-UGI-NLS-P2A-EGFP or control pCAG-P2A-EGFP constructs with various gRNAs as described below. Cells were sorted for top 5% GFP or all GFP + cells based on FITC signal. RNA-seq was performed to measure transcriptional changes associated with different constructs and guides.
Publication Title
Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors.
Sample Metadata Fields
Cell line, Treatment, Subject
View Samples- Homo sapiens
- 9 Downloadable Samples
- NextSeq 500
Description
Base Editing has been touted the most intelligent and precise application of the CRISPR platform so far, merging the simplicity of RNA-guided nucleases with deaminases that allow for the programmable generation of single base substitutions - without introduction of double-strand breaks. Even though the two-component system has been expected to cause off-target substitutions, studies involving cytosine base editors (CBEs) showed that in most cases, relatively few single base off-targets could be detected on DNA. We introduce the concept of multi-dimensional off-targeting, presenting an extensive amount of RNA cytidines being edited by DNA base editors. Epitranscriptomic off-target effects affected different cell lines and were independent of the guide RNAs used, suggesting Cas9-independent activity of the cytidine deaminase rAPOBEC1 on single-stranded RNA. With the help of protein engineering, we developed CBE variants with massively reduced inadvertent mutation of RNA that preserve and enhance DNA base editing capabilities. Overall design: HEK293T cells were transfected with pCAG-BE3-P2A-EGFP or variants thereof or control pCAG-nCas9(D10A)-UGI-NLS-P2A-EGFP or control pCAG-P2A-EGFP constructs with various gRNAs as described below. Cells were sorted for top 5% GFP or all GFP + cells based on FITC signal. RNA-seq was performed to measure transcriptional changes associated with different constructs and guides.
Publication Title
Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors.
Sample Metadata Fields
Cell line, Treatment, Subject
View Samples- Homo sapiens
- 2 Downloadable Samples
- Illumina HiSeq 2500
Description
Base Editing has been touted the most intelligent and precise application of the CRISPR platform so far, merging the simplicity of RNA-guided nucleases with deaminases that allow for the programmable generation of single base substitutions - without introduction of double-strand breaks. Even though the two-component system has been expected to cause off-target substitutions, studies involving cytosine base editors (CBEs) showed that in most cases, relatively few single base off-targets could be detected on DNA. We introduce the concept of multi-dimensional off-targeting, presenting an extensive amount of RNA cytidines being edited by DNA base editors. Epitranscriptomic off-target effects affected different cell lines and were independent of the guide RNAs used, suggesting Cas9-independent activity of the cytidine deaminase rAPOBEC1 on single-stranded RNA. With the help of protein engineering, we developed CBE variants with massively reduced inadvertent mutation of RNA that preserve and enhance DNA base editing capabilities. Overall design: HEK293T or HepG2 cells were transfected with P2A-EGFP. Cells were sorted for top 5% GFP based on FITC signal. RNA-seq was performed to measure transcriptional changes associated with different constructs.
Publication Title
Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors.
Sample Metadata Fields
Specimen part, Treatment, Subject
View Samples- Homo sapiens
- 4 Downloadable Samples
Affymetrix Human Exon 1.0 ST Array [transcript (gene) version (huex10st)
Description
Epigenetic events, including covalent post-translational modification of histones, have frequently been demonstrated to play critical roles in tumor development and progression. The transcriptional coactivator, p300/CBP, possesses both histone acetyltransferase (HAT) activity as well as scaffolding properties that directly influence transcriptional activation of targeted genes. We have used a recently reported small molecule inhibitor of p300 HAT activity, C646, to explore the specific contribution of p300/CBP HAT activity to tumor development and progression. We find that C646 inhibits the growth of lineage-specific tumor cell lines including human melanomas through direct transcriptional regulation of cell cycle regulatory proteins. Further evaluation of the p300 HAT transcriptome in human melanoma cells using comprehensive gene expression profiling reveals that p300 HAT activity globally promotes cell cycle progression, nucleosome assembly, and the DNA damage checkpoint through direct transcriptional regulatory mechanisms. Additionally, C646 promotes sensitivity to DNA damaging agents leading to enhanced apoptosis of melanoma cells following combination treatment with cisplatin. Together our data suggest that p300 HAT activity regulates critical growth regulatory pathways in tumors and may serve as a novel therapeutic target for melanoma and other malignancies by promoting cellular responses to DNA damaging agents.
Publication Title
Selective inhibition of p300 HAT blocks cell cycle progression, induces cellular senescence, and inhibits the DNA damage response in melanoma cells.
Sample Metadata Fields
Cell line, Treatment, Time
View Samples- Homo sapiens
- 28 Downloadable Samples
- NextSeq 500
Description
In vitro cultured CD34+ derived erythroblasts were sorted using surface markers and processed using RNA-seq Overall design: Biological replicates (3 or 4 per population) were processed across 2-3 biological donors for 8 sorted populations for RNA-seq
Publication Title
Transcriptional States and Chromatin Accessibility Underlying Human Erythropoiesis.
Sample Metadata Fields
Subject
View Samples- Homo sapiens
- 7 Downloadable Samples
- NextSeq 500
Description
HUDEP-2 cells were lentivirally infected with CRISPRi constructs using a nontargeting guide or guides targeting an enhancer in the TMCC2 locus Overall design: Whole transcriptome libraries were sequenced for three replicates of non-targeting gRNA and two replicates each for two different gRNA targeting a regulatory region upstream of the TMCC2 erythroid-specific isoform
Publication Title
Transcriptional States and Chromatin Accessibility Underlying Human Erythropoiesis.
Sample Metadata Fields
Cell line, Subject
View Samples- Mus musculus
- 10 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
Epithelial tip progenitor cells are an important epithelial progenitor population in the developing lung. At early stages of development they produce SOX2+ bronchiolar progenitor cells. At later stages of embryonic lung development they produce SOX2- alveolar progenitor cells.
Publication Title
Lung epithelial tip progenitors integrate glucocorticoid- and STAT3-mediated signals to control progeny fate.
Sample Metadata Fields
Specimen part
View Samples- Danio rerio
- 246 Downloadable Samples
- NextSeq 500
Description
Recent advances in single-cell transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. Here, we utilized massively parallel single-cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow, constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally-distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner, our approach was useful in characterizing immune cell deficiencies within prkdcD3612fs, il2rgaY91fs and double homozygous mutant fish, identifying blood cell losses in T, B, and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types including two classes of natural killer immune cells, classically-defined and erythroid-primed hematopoietic stem and progenitor cells, mucin secreting kidney cells, and kidney stem/progenitor cells. In total, our work provides the first comprehensive single cell transcriptomic analysis of kidney and marrow cells in the adult zebrafish. Overall design: The goal of our study is to establish the transcriptional profiles of hematopoietic and kidney cell lineages residing in the zebrafish whole kidney marrow. Firstly, we performed single-cell RNA sequencing by a modified Smart-seq2 protocol on sorted single cells from fluorescent transgenic zebrafish lines, which label distinct blood cell types (n = 246 cells total). Secondly, we utilized droplet-based single-cell RNA sequencing (inDrop) to investigate unmarked, comprehensive hematopoietic lineage structure within wild-type, casper-strain zebrafish (N=3 animals, n=3,782 cells total). From this, we identified ten distinct hematopoietic groups of blood and immune identities. Thirdly, we confirmed blood lineage interpretations by comparing hematopoietic lineages within wild-type fish with mutant zebrafish with known immunodeficiencies, including prkdc(D3612fs) (N=3 animals, n=3,201 cells), il2rga(Y91fs) (N=2 animals, n=2,068 cells) and prkdc(D3612fs), il2rga(Y91fs) double compound mutant fish (N=2 animals, n=2,276 cells). Lastly, we identified seven structural and functional cell lineages of kidney identities in the whole kidney marrow (n=1,699 kidney cells).
Publication Title
Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing.
Sample Metadata Fields
Specimen part, Subject
View Samples