المؤلفون: Phong Nguyen, Ines Hellmann, Lucas E. Wange, Johannes W. Bagnoli, Daniel Richter, Irmela Jeremias, Christoph Ziegenhain, Wolfgang Enard, Johanna Geuder, Beate Vieth, Binje Vick, Aleksandar Janjic

المصدر: Genome Biol. 23:88 (2022)

مصطلحات موضوعية: Protocol (science), Profit (accounting), Base Sequence, Sequence Analysis, RNA, Computer science, genetic processes, RNA, computer.software_genre, Genomics, Power Analysis, Rna-seq, Transcriptomics, DNA sequencing, Prime (order theory), Statistical power, Exome Sequencing, natural sciences, RNA extraction, Data mining, computer, health care economics and organizations, Gene Library

الوصف: With the advent of Next Generation Sequencing, RNA-sequencing (RNA-seq) has become the major method for quantitative gene expression analysis. Reducing library costs by early barcoding has propelled single-cell RNA-seq, but has not yet caught on for bulk RNA-seq. Here, we optimized and validated a bulk RNA-seq method we call prime-seq. We show that with respect to library complexity, measurement accuracy, and statistical power it performs equivalent to TruSeq, a standard bulk RNA-seq method, but is four-fold more cost-efficient due to almost 50-fold cheaper library costs. We also validate a direct RNA isolation step that further improves cost and time-efficiency, show that intronic reads are derived from RNA, validate that prime-seq performs optimal with only 1,000 cells as input, and calculate that prime-seq is the most cost-efficient bulk RNA-seq method currently available. We discuss why many labs would profit from a cost-efficient early barcoding RNA-seq protocol and argue that prime-seq is well suited for setting up such a protocol as it is well validated, well documented, and requires no specialized equipment.

وصف الملف: application/pdf

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::8fb9e8929a33ba0ce58e8fb3d5aaed50Test
https://doi.org/10.1186/s13059-022-02660-8Test

المؤلفون: Lucas E. Wange, Johannes W. Bagnoli, Wolfgang Enard, Johanna Geuder, Aleksandar Janjic, Swati Parekh, Beate Vieth, Ines Hellmann, Christoph Ziegenhain

المصدر: Nature Communications, Vol 9, Iss 1, Pp 1-8 (2018)
Nature Communications

مصطلحات موضوعية: 0301 basic medicine, Sequence analysis, Computer science, Science, genetic processes, Cell, General Physics and Astronomy, Computational biology, Article, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Library Protocol, Single-cell analysis, Complementary DNA, medicine, natural sciences, lcsh:Science, Polymerase, Multidisciplinary, Base Sequence, biology, Sequence Analysis, RNA, High-Throughput Nucleotide Sequencing, RNA, General Chemistry, 030104 developmental biology, medicine.anatomical_structure, biology.protein, lcsh:Q, Research questions, Single-Cell Analysis, Software

الوصف: Single-cell RNA sequencing (scRNA-seq) has emerged as a central genome-wide method to characterize cellular identities and processes. Consequently, improving its sensitivity, flexibility, and cost-efficiency can advance many research questions. Among the flexible plate-based methods, single-cell RNA barcoding and sequencing (SCRB-seq) is highly sensitive and efficient. Here, we systematically evaluate experimental conditions of this protocol and find that adding polyethylene glycol considerably increases sensitivity by enhancing cDNA synthesis. Furthermore, using Terra polymerase increases efficiency due to a more even cDNA amplification that requires less sequencing of libraries. We combined these and other improvements to develop a scRNA-seq library protocol we call molecular crowding SCRB-seq (mcSCRB-seq), which we show to be one of the most sensitive, efficient, and flexible scRNA-seq methods to date.
Single-cell RNA-barcoding and sequencing is an efficient, genome-wide method to characterize cellular identities. Here the authors systematically evaluate the protocol and develop molecular crowding SCRB-seq with improved sensitivity and cost-efficiency.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::713e8f80a7592fca6db998d81d92a0c4Test
https://doaj.org/article/4a131646ab114fc18a5468c079161e41Test

المؤلفون: Aleksandar Janjic, Swati Parekh, Beate Vieth, Wolfgang Enard, Christoph Ziegenhain, Ines Hellmann, Johannes W. Bagnoli, Johanna Geuder, Lucas E. Wange

مصطلحات موضوعية: Genetics, biology, genetic processes, Cell, RNA, Genomics, Computational biology, medicine.anatomical_structure, Library Protocol, Complementary DNA, medicine, biology.protein, natural sciences, Sensitivity (control systems), Enhancer, Polymerase

الوصف: SummarySingle-cell RNA sequencing (scRNA-seq) has emerged as the central genome-wide method to characterize cellular identities and processes. While performance of scRNA-seq methods is improving, an optimum in terms of sensitivity, cost-efficiency and flexibility has not yet been reached. Among the flexible plate-based methods “Single-Cell RNA-Barcoding and Sequencing” (SCRB-seq) is one of the most sensitive and efficient ones. Based on this protocol, we systematically evaluated experimental conditions such as reverse transcriptases, reaction enhancers and PCR polymerases. We find that adding polyethylene glycol considerably increases sensitivity by enhancing cDNA synthesis. Furthermore, using Terra polymerase increases efficiency due to a more even cDNA amplification that requires less sequencing of libraries. We combined these and other improvements to a new scRNA-seq library protocol we call “molecular crowding SCRB-seq” (mcSCRB-seq), which we show to be the most sensitive and one of the most efficient and flexible scRNA-seq methods to date.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::79905c0191d07e001b3653947ff0af94Test
https://doi.org/10.1101/188367Test