Scalable Transcriptome Preparation for Massive Parallel Sequencing

Stranneheim, Henrik; Werne, Beata; Sherwood, Ellen; Lundeberg, Joakim

doi:10.1371/journal.pone.0021910

Cited by 19 publications

(21 citation statements)

References 13 publications

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“…For gene profiling, total RNA from confluent cultures of the two fibroblast pairs were analyzed using the Illumina HiSeq technology, identifying 53,196 expressed mRNAs and noncoding regulatory RNAs 19. To identify the differentially expressed transcripts in relation to the inhibitory phenotype, we selected the genes whose expression profile showed the following features:…”

Section: Resultsmentioning

confidence: 99%

Impaired podosome formation and invasive migration of macrophages from patients with a PSTPIP1 mutation and PAPA syndrome

Cortesio¹,

Wernimont²,

Kastner³

et al. 2010

Arthritis & Rheumatism

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Normal human and murine fibroblasts can inhibit proliferation of tumor cells when co‐cultured in vitro. The inhibitory capacity varies depending on the donor and the site of origin of the fibroblast. It requires direct cell‐to‐cell contact and is not transferable with supernatant. Here, we show that effective inhibition also requires the formation of a morphologically intact fibroblast monolayer before the seeding of the tumor cells. Interference with the formation of the monolayer impairs the inhibition. Subclones of TERT‐immortalized fibroblasts were selected on the basis of differences in the growth pattern and related inhibitory activity. Whereas the well‐organized “whirly” (WH) growth pattern was associated with strong inhibition, the disorganized “crossy” (CR) growth pattern was linked to reduced inhibition. Time lapse imaging of tumor‐fibroblast co‐cultures using extended field live cell microscopy revealed that fibroblast monolayers with growth inhibitory capacity also reduced the motility of the tumor cells whereas noninhibitory monolayers had no effect on tumor cell motility. Gene expression pattern of two isogenic pairs of fibroblasts, WH and CR subclones of the TERT immortalized line (inhibitory, and less inhibitory subsequently) and freshly explanted skin (inhibitory) and hernia (noninhibitory) fibroblasts derived from the same patient, identified a set of genes that co‐segregated with the inhibitory phenotype. This suggests that our model system may reveal molecular mechanisms involved in contact‐mediated microenvironmental surveillance that may protect the organism from the outgrowth of disseminated tumor cells.

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Section: Resultsmentioning

confidence: 99%

Impaired podosome formation and invasive migration of macrophages from patients with a PSTPIP1 mutation and PAPA syndrome

Cortesio¹,

Wernimont²,

Kastner³

et al. 2010

Arthritis & Rheumatism

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show abstract

“…The RNA extracts were stored at -80°C. Each RNA sample was bar-coded and prepared according to Illumina mRNA-seq sample preparation and kit with the automated platform previously described [18]. The barcoded libraries were pooled together in pairs at equal concentrations and clustered on a cBot cluster-generation system using the Illumina HiSeq single-read cluster generation kit according to the protocol from the manufacturer.…”

Section: Methodsmentioning

confidence: 99%

Comprehensive analysis of the genome transcriptome and proteome landscapes of three tumor cell lines

et al. 2012

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We here present a comparative genome, transcriptome and functional network analysis of three human cancer cell lines (A431, U251MG and U2OS), and investigate their relation to protein expression. Gene copy numbers significantly influenced corresponding transcript levels; their effect on protein levels was less pronounced. We focused on genes with altered mRNA and/or protein levels to identify those active in tumor maintenance. We provide comprehensive information for the three genomes and demonstrate the advantage of integrative analysis for identifying tumor-related genes amidst numerous background mutations by relating genomic variation to expression/protein abundance data and use gene networks to reveal implicated pathways.

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“…This has been achieved by setting up library preparation protocols on robotic workstations9101112131415. It has previously been shown that a 12-channel liquid handling robot can increase sample preparation throughput by up to six times9.…”

mentioning

confidence: 99%

An automated approach to prepare tissue-derived spatially barcoded RNA-sequencing libraries

Jemt

Salmén

Lundmark

et al. 2016

Sci Rep

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Sequencing the nucleic acid content of individual cells or specific biological samples is becoming increasingly common. This drives the need for robust, scalable and automated library preparation protocols. Furthermore, an increased understanding of tissue heterogeneity has lead to the development of several unique sequencing protocols that aim to retain or infer spatial context. In this study, a protocol for retaining spatial information of transcripts has been adapted to run on a robotic workstation. The method spatial transcriptomics is evaluated in terms of robustness and variability through the preparation of reference RNA, as well as through preparation and sequencing of six replicate sections of a gingival tissue biopsy from a patient with periodontitis. The results are reduced technical variability between replicates and a higher throughput, processing four times more samples with less than a third of the hands on time, compared to the standard protocol.

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Scalable Transcriptome Preparation for Massive Parallel Sequencing

Cited by 19 publications

References 13 publications

Impaired podosome formation and invasive migration of macrophages from patients with a PSTPIP1 mutation and PAPA syndrome

Impaired podosome formation and invasive migration of macrophages from patients with a PSTPIP1 mutation and PAPA syndrome

Comprehensive analysis of the genome transcriptome and proteome landscapes of three tumor cell lines

An automated approach to prepare tissue-derived spatially barcoded RNA-sequencing libraries

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