Stranded RNA-Sequencing Method_The Broad Institute v1

2021 ◽  
Author(s):  
Broad Institute, Genomics Platform
Keyword(s):  
Rna Sequencing ◽  
Human Tumor ◽  
Pilot Project ◽  
Broad Institute

Here we describe the TruSeq Strand Specific Large Insert Method followed by The Broad Genomics Platform to sequence bulk RNA from human tumor biospecimens for the Human Tumor Atlas Pilot Project (HTAPP). During the years, modifications were introduced to the method resulting in slightly different versions employed. Below we provide the description of the three different versions used on HTAPP samples (from newer to older) highlighting their differences in bold lettering.

scholarly journals High-throughput RNA sequencing of paraformaldehyde-fixed single cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hoang Van Phan ◽  
Michiel van Gent ◽  
Nir Drayman ◽  
Anindita Basu ◽  
Michaela U. Gack ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
High Throughput ◽  
Single Cells ◽  
Human Tumor ◽  
Viral Reactivation ◽  
Rna Integrity ◽  
High Throughput Method ◽  
Tumor Virus ◽  
Cell Subpopulations

AbstractSingle-cell transcriptomic studies that require intracellular protein staining, rare cell sorting, or inactivation of infectious pathogens are severely limited. This is because current high-throughput single-cell RNA sequencing methods are either incompatible with or necessitate laborious sample preprocessing for paraformaldehyde treatment, a common tissue and cell fixation and preservation technique. Here we present FD-seq (Fixed Droplet RNA sequencing), a high-throughput method for droplet-based RNA sequencing of paraformaldehyde-fixed, permeabilized and sorted single cells. We show that FD-seq preserves the RNA integrity and relative gene expression levels after fixation and permeabilization. Furthermore, FD-seq can detect a higher number of genes and transcripts than methanol fixation. We first apply FD-seq to analyze a rare subpopulation of cells supporting lytic reactivation of the human tumor virus KSHV, and identify TMEM119 as a potential host factor that mediates viral reactivation. Second, we find that infection with the human betacoronavirus OC43 leads to upregulation of pro-inflammatory pathways in cells that are exposed to the virus but fail to express high levels of viral genes. FD-seq thus enables integrating phenotypic with transcriptomic information in rare cell subpopulations, and preserving and inactivating pathogenic samples.

scholarly journals Further investigation of the potential anti-neoplastic, anti-inflammatory and immunomodulatory actions of phenoxybenzamine using the Broad Institute CLUE platform

2019 ◽  
Author(s):  
Mario A. Inchiosa
Keyword(s):  
Gene Expression ◽  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitors ◽  
Glycogen Synthase ◽  
Human Tumor ◽  
Gene Expression Signature ◽  
Broad Institute ◽  
Adrenergic Antagonist ◽  
Gene Expression Signatures ◽  
Anti Inflammatory

AbstractPrevious clinical studies with the FDA-approved alpha-adrenergic antagonist, phenoxybenzamine, showed apparent efficacy to reverse the symptoms and disabilities of the neuropathic condition, Complex Regional Pain Syndrome; also, the anatomic spread and intensity of this syndrome has a proliferative character and it was proposed that phenoxybenzamine may have an anti-inflammatory, immunomodulatory mode of action. A previous study gave evidence that phenoxybenzamine had anti-proliferative activity in suppression of growth in several human tumor cell cultures. The same report demonstrated that the drug possessed significant histone deacetylase inhibitory activity. Utilizing the Harvard/Massachusetts Institute of Technology Broad Institute genomic database, CLUE, the present study suggests that the gene expression signature of phenoxybenzamine in malignant cell lines is consistent with anti-inflammatory/immunomodulatory activity and suppression of tumor expansion by several possible mechanisms of action. Of particular note, phenoxybenzamine demonstrated signatures that were highly similar to those with glucocorticoid agonist activity. Also, gene expression signatures of phenoxbenzamine were consistent with several agents in each case that were known to suppress tumor proliferation, notably, protein kinase C inhibitors, Heat Shock Protein inhibitors, epidermal growth factor receptor inhibitors, and glycogen synthase kinase inhibitors. Searches in CLUE also confirmed the earlier observations of strong similarities between gene expression signatures of phenoxybenzamine and several histone deacetylase inhibitors.

scholarly journals RNA Sequencing-Based Identification of Ganglioside GD2-Positive Cancer Phenotype

Biomedicines ◽  
2020 ◽  
Vol 8 (6) ◽  
pp. 142 ◽  
Author(s):  
Maxim Sorokin ◽  
Irina Kholodenko ◽  
Daniel Kalinovsky ◽  
Tatyana Shamanskaya ◽  
Igor Doronin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
Human Tumor ◽  
Gene Expression Signature ◽  
Sequencing Data ◽  
Biopsy Material ◽  
Ganglioside Gd2 ◽  
Expression Signature ◽  
Ganglioside Biosynthesis ◽  
Tumor Phenotypes

The tumor-associated ganglioside GD2 represents an attractive target for cancer immunotherapy. GD2-positive tumors are more responsive to such targeted therapy, and new methods are needed for the screening of GD2 molecular tumor phenotypes. In this work, we built a gene expression-based binary classifier predicting the GD2-positive tumor phenotypes. To this end, we compared RNA sequencing data from human tumor biopsy material from experimental samples and public databases as well as from GD2-positive and GD2-negative cancer cell lines, for expression levels of genes encoding enzymes involved in ganglioside biosynthesis. We identified a 2-gene expression signature combining ganglioside synthase genes ST8SIA1 and B4GALNT1 that serves as a more efficient predictor of GD2-positive phenotype (Matthews Correlation Coefficient (MCC) 0.32, 0.88, and 0.98 in three independent comparisons) compared to the individual ganglioside biosynthesis genes (MCC 0.02–0.32, 0.1–0.75, and 0.04–1 for the same independent comparisons). No individual gene showed a higher MCC score than the expression signature MCC score in two or more comparisons. Our diagnostic approach can hopefully be applied for pan-cancer prediction of GD2 phenotypes using gene expression data.

scholarly journals Fixed single-cell RNA sequencing for understanding virus infection and host response

2020 ◽  
Author(s):  
Hoang Van Phan ◽  
Michiel van Gent ◽  
Nir Drayman ◽  
Anindita Basu ◽  
Michaela U. Gack ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
High Throughput ◽  
Single Cells ◽  
Human Tumor ◽  
Tissue Cell ◽  
Pathogen Inactivation ◽  
Single Cell Rna Sequencing ◽  
Sequencing Studies ◽  
High Level

ABSTRACTSingle-cell RNA sequencing studies requiring intracellular protein staining, rare-cell sorting, or pathogen inactivation are severely limited because current high-throughput methods are incompatible with paraformaldehyde treatment, a very common and simple tissue/cell fixation and preservation technique. Here we present FD-seq, a high-throughput method for droplet-based RNA sequencing of paraformaldehyde-fixed, stained and sorted single-cells. We used FD-seq to address two important questions in virology. First, by analyzing a rare population of cells supporting lytic reactivation of the human tumor virus KSHV, we identified TMEM119 as a host factor that mediates reactivation. Second, we studied the transcriptome of lung cells infected with the coronavirus OC43, which causes the common cold and also serves as a safer model pathogen for SARS-CoV-2. We found that pro-inflammatory pathways are primarily upregulated in abortively-infected or uninfected bystander cells, which are exposed to the virus but fail to express high level of viral genes. FD-seq is suitable for characterizing rare cell populations of interest, for studying high-containment biological samples after inactivation, and for integrating intracellular phenotypic with transcriptomic information.

Targeted Expansion Sequencing Protocols v2

2021 ◽  
Author(s):  
Anubhav Sinha ◽  
Yi Cui ◽  
Shahar Alon ◽  
Fei Chen ◽  
Asmamaw T. Wassie ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Human Tumor ◽  
Metastatic Breast ◽  
Pilot Project ◽  
Probe Design ◽  
Tissue Preparation ◽  
Padlock Probe ◽  
Library Preparation ◽  
Key Steps

This protocol collection accompanies accompanies Expansion Sequencing (ExSeq), covering the four key steps of a targeted Expansion Sequencing (targeted ExSeq) experiment: (1) Padlock probe design; (2) tissue preparation and expansion; (3) library preparation; and (4) in situ sequencing with the Illumina chemistry. For further details, consult the relevant protocols within the collection. These protocols were used to profile human metastatic breast cancer biopsies as a part of the Human Tumor Atlas Pilot Project (HTAPP).

Exome ICE sequencing Methods_The Broad Institute v1

2021 ◽  
Author(s):  
Broad Institute, Genomics Platform
Keyword(s):  
Genomic Dna ◽  
Pilot Project ◽  
Broad Institute

Here we describe the Exome ICE Method followed by The Broad Genomics Platform for sequencing genomic DNA from human biospecimens for the Human Atlas Pilot Project (HTAPP). During the years, modifications were introduced to the method resulting in slightly different versions employed. Below we provide the description of the three different versions used on HTAPP samples (from newer to older) highlighting their differences in bold lettering.

Targeted Expansion Sequencing Protocols v3

2021 ◽  
Author(s):  
Anubhav Sinha ◽  
Yi Cui ◽  
Shahar Alon ◽  
Fei Chen ◽  
Asmamaw T. Wassie ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Human Tumor ◽  
Metastatic Breast ◽  
Pilot Project ◽  
Probe Design ◽  
Tissue Preparation ◽  
Padlock Probe ◽  
Library Preparation ◽  
Key Steps

This protocol collection accompanies accompanies Expansion Sequencing (ExSeq), covering the four key steps of a targeted Expansion Sequencing (targeted ExSeq) experiment: (1) Padlock probe design; (2) tissue preparation and expansion; (3) library preparation; and (4) in situ sequencing with the Illumina chemistry. For further details, consult the relevant protocols within the collection. These protocols were used to profile human metastatic breast cancer biopsies as a part of the Human Tumor Atlas Pilot Project (HTAPP).

Transcriptome Capture Sequencing Method_The Broad Institute v1

2021 ◽  
Author(s):  
Broad Institute, Genomics Platform
Keyword(s):  
Pilot Project ◽  
Broad Institute ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Capture Method ◽  
Formalin Fixed ◽  
Capture Sequencing

Here we describe the Transcriptome Capture Method followed by The Broad Genomics Platform for sequencing bulk RNA from human biospecimens for the Human Atlas Pilot Project (HTAPP). This protocol is usually utilized for Formalin Fixed Paraffin Embedded (FFPE) samples, although it can also be used for non-FFPE samples, as was the case for some HTAPP samples.

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

1982 ◽  
Vol 40 ◽  
pp. 210-211
Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman
Keyword(s):  
Cultured Cells ◽  
Human Tumor ◽  
Cell Type ◽  
Tumor Mass ◽  
Initial Cell ◽  
Cloning Assay ◽  
Human Tumor Cloning ◽  
The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

Virus-Like Particles in a Human Breast Cancer: Structural Similarity to Previously Reported Particles

1973 ◽  
Vol 31 ◽  
pp. 378-379
Author(s):  
G. Kasnic ◽  
S. E. Stewart ◽  
C. Urbanski
Keyword(s):  
Breast Cancer ◽  
Biological Activity ◽  
Human Breast Cancer ◽  
Osteogenic Sarcoma ◽  
Human Tumor ◽  
Structural Similarity ◽  
Cell Tumor ◽  
Human Breast ◽  
Human Tumor Cell ◽  
Type Particle

We have reported the maturation of an intracisternal A-type particle in murine plasma cell tumor cultures and three human tumor cell cultures (rhabdomyosarcoma, lung adenocarcinoma, and osteogenic sarcoma) after IUDR-DMSO activation. In all of these studies the A-type particle seems to develop into a form with an electron dense nucleoid, presumably mature, which is also intracisternal. A similar intracisternal A-type particle has been described in leukemic guinea pigs. Although no biological activity has yet been demonstrated for these particles, on morphologic grounds, and by the manner in which they develop within the cell, they may represent members of the same family of viruses.

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