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Method Article
Eric Charpentier
CHU Nantes, Inserm, CNRS, SFR Santé, Inserm UMS016, CNRS UMS 3556, Université de Nantes, 44000, Nantes, France
Corresponding Author
ORCiD: https://orcid.org/0000-0002-8571-7603
Marine Cornec
CHU Nantes, Inserm, CNRS, SFR Santé, Inserm UMS016, CNRS UMS 3556, Université de Nantes, 44000, Nantes, France
Corresponding Author
ORCiD: https://orcid.org/0000-0001-9102-753X
Stéphanie Bonnaud
Université de Nantes, CHU Nantes, Inserm, CNRS, SFR Santé, Inserm UMS 016, CNRS UMS 3556, F-44000 Nantes, France
Corresponding Author
Audrey Bihouée
Université de Nantes, CHU Nantes, Inserm, CNRS, SFR Santé, Inserm UMS 016, CNRS UMS 3556, F-44000 Nantes, France
Corresponding Author
ORCiD: https://orcid.org/0000-0002-8689-2083
License:
This work is licensed under a CC BY 4.0 License. Read Full License
3’seq-RNA Profiling (3’SRP) approach is based on multiplexing samples and molecular indexing mRNA in order to drive genome-wide transcriptional profiling at reasonable cost in comparison to standard RNA-sequencing. The protocol is performed according to the 3′-digital gene expression (3′-DGE) approach developed by the Broad institute. The libraries are prepared from small amounts of total RNA where the mRNA poly(A) tails are tagged with universal adapters, well-specific barcodes and unique molecular identifiers (UMIs). We have improved the fragmentation step by implementing tagmentation based on the activity of a bead-linked transposome. This technique allows sample multiplexing on 96-well plates. Libraries are then sequenced using standard procedures, e.g. on Hiseq2500 or NovaSeq 6000 SP Flow Cells. We have developed a snakemake pipeline including every analysis step from raw fastq de-multiplexing to functional annotation of the differentially expressed genes, producing a complete HTML report for end-user.
Keywords
transcriptome,expression,sequencing,pipeline,snakemake
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Associated Publications
Parallel derivation of isogenic human primed and naive induced pluripotent stem cells
by Stéphanie Kilens, , Dimitri Meistermann, +28
Nature Communications (18 January, 2018)
Ex Vivo Expanded Human Non-Cytotoxic CD8+CD45RClow/− Tregs Efficiently Delay Skin Graft Rejection and GVHD in Humanized Mice
by Séverine Bézie, Dimitri Meistermann, Laetitia Boucault, +11
Frontiers in Immunology (30 January, 2018)
An intermediate level of CD161 expression defines a novel activated, inflammatory, and pathogenic subset of CD8 + T cells involved in multiple sclerosis
by Bryan Nicol, Marion Salou, Isabel Vogel, +19
Journal of Autoimmunity (18 October, 2017)
Transient antibody targeting of CD45RC induces transplant tolerance and potent antigen-specific regulatory T cells
by Elodie Picarda, Séverine Bézie, Laetitia Boucault, +10
JCI Insight (08 February, 2017)
Characterization of Glyceollins as Novel Aryl Hydrocarbon Receptor Ligands and Their Role in Cell Migration
by Thu Ha Pham, Sylvain Lecomte, Remy Le Guevel, +7
International Journal of Molecular Sciences (19 February, 2020)
Maternal Protein Restriction in Rats Alters the Expression of Genes Involved in Mitochondrial Metabolism and Epitranscriptomics in Fetal Hypothalamus
by Morgane Frapin, Simon Guignard, Dimitri Meistermann, +5
Nutrients (20 May, 2020)
Dual targeting of BCL2 and MCL1 rescues myeloma cells resistant to BCL2 and MCL1 inhibitors associated with the formation of BAX/BAK hetero-complexes
by Carolane Seiller, Sophie Maiga, Cyrille Touzeau, +8
Cell Death & Disease (05 May, 2020)
Induction of Human Trophoblast Stem Cells from Somatic Cells and Pluripotent Stem Cells
by Gaël Castel, Dimitri Meistermann, Betty Bretin, +24
Cell Reports (24 November, 2020)
NOTO Transcription Factor Directs Human Induced Pluripotent Stem Cell-Derived Mesendoderm Progenitors to a Notochordal Fate
by Pauline Colombier, Boris Halgand, Claire Chédeville, +8
Cells (25 February, 2020)
Human model of IRX5 mutations reveals key role for this transcription factor in ventricular conduction
by Zeina R Al Sayed, Robin Canac, Bastien Cimarosti, +19
Cardiovascular Research (28 August, 2020)
STING-dependent paracriny shapes apoptotic priming of breast tumors in response to anti-mitotic treatment
by Steven Lohard, Nathalie Bourgeois, Laurent Maillet, +15
Nature Communications (14 January, 2020)
Urine-derived cells provide a readily accessible cell type for feeder-free mRNA reprogramming
by A. Gaignerie, N. Lefort, M. Rousselle, +13
Scientific Reports (19 September, 2018)
An open repository of community-contributed protocols sponsored by Nature Research.
Learn more about Protocol Exchange
Method Article
Eric Charpentier
CHU Nantes, Inserm, CNRS, SFR Santé, Inserm UMS016, CNRS UMS 3556, Université de Nantes, 44000, Nantes, France
Corresponding Author
ORCiD: https://orcid.org/0000-0002-8571-7603
Marine Cornec
CHU Nantes, Inserm, CNRS, SFR Santé, Inserm UMS016, CNRS UMS 3556, Université de Nantes, 44000, Nantes, France
Corresponding Author
ORCiD: https://orcid.org/0000-0001-9102-753X
Stéphanie Bonnaud
Université de Nantes, CHU Nantes, Inserm, CNRS, SFR Santé, Inserm UMS 016, CNRS UMS 3556, F-44000 Nantes, France
Corresponding Author
Audrey Bihouée
Université de Nantes, CHU Nantes, Inserm, CNRS, SFR Santé, Inserm UMS 016, CNRS UMS 3556, F-44000 Nantes, France
Corresponding Author
ORCiD: https://orcid.org/0000-0002-8689-2083
Version History
CURRENT STATUS: Posted
Version 1
Posted 28 Jan, 2021
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
3’seq-RNA Profiling (3’SRP) approach is based on multiplexing samples and molecular indexing mRNA in order to drive genome-wide transcriptional profiling at reasonable cost in comparison to standard RNA-sequencing. The protocol is performed according to the 3′-digital gene expression (3′-DGE) approach developed by the Broad institute. The libraries are prepared from small amounts of total RNA where the mRNA poly(A) tails are tagged with universal adapters, well-specific barcodes and unique molecular identifiers (UMIs). We have improved the fragmentation step by implementing tagmentation based on the activity of a bead-linked transposome. This technique allows sample multiplexing on 96-well plates. Libraries are then sequenced using standard procedures, e.g. on Hiseq2500 or NovaSeq 6000 SP Flow Cells. We have developed a snakemake pipeline including every analysis step from raw fastq de-multiplexing to functional annotation of the differentially expressed genes, producing a complete HTML report for end-user.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Loading...
Associated Publications
Parallel derivation of isogenic human primed and naive induced pluripotent stem cells
by Stéphanie Kilens, , Dimitri Meistermann, +28
Nature Communications (18 January, 2018)
Ex Vivo Expanded Human Non-Cytotoxic CD8+CD45RClow/− Tregs Efficiently Delay Skin Graft Rejection and GVHD in Humanized Mice
by Séverine Bézie, Dimitri Meistermann, Laetitia Boucault, +11
Frontiers in Immunology (30 January, 2018)
An intermediate level of CD161 expression defines a novel activated, inflammatory, and pathogenic subset of CD8 + T cells involved in multiple sclerosis
by Bryan Nicol, Marion Salou, Isabel Vogel, +19
Journal of Autoimmunity (18 October, 2017)
Transient antibody targeting of CD45RC induces transplant tolerance and potent antigen-specific regulatory T cells
by Elodie Picarda, Séverine Bézie, Laetitia Boucault, +10
JCI Insight (08 February, 2017)
Characterization of Glyceollins as Novel Aryl Hydrocarbon Receptor Ligands and Their Role in Cell Migration
by Thu Ha Pham, Sylvain Lecomte, Remy Le Guevel, +7
International Journal of Molecular Sciences (19 February, 2020)
Maternal Protein Restriction in Rats Alters the Expression of Genes Involved in Mitochondrial Metabolism and Epitranscriptomics in Fetal Hypothalamus
by Morgane Frapin, Simon Guignard, Dimitri Meistermann, +5
Nutrients (20 May, 2020)
Dual targeting of BCL2 and MCL1 rescues myeloma cells resistant to BCL2 and MCL1 inhibitors associated with the formation of BAX/BAK hetero-complexes
by Carolane Seiller, Sophie Maiga, Cyrille Touzeau, +8
Cell Death & Disease (05 May, 2020)
Induction of Human Trophoblast Stem Cells from Somatic Cells and Pluripotent Stem Cells
by Gaël Castel, Dimitri Meistermann, Betty Bretin, +24
Cell Reports (24 November, 2020)
NOTO Transcription Factor Directs Human Induced Pluripotent Stem Cell-Derived Mesendoderm Progenitors to a Notochordal Fate
by Pauline Colombier, Boris Halgand, Claire Chédeville, +8
Cells (25 February, 2020)
Human model of IRX5 mutations reveals key role for this transcription factor in ventricular conduction
by Zeina R Al Sayed, Robin Canac, Bastien Cimarosti, +19
Cardiovascular Research (28 August, 2020)
STING-dependent paracriny shapes apoptotic priming of breast tumors in response to anti-mitotic treatment
by Steven Lohard, Nathalie Bourgeois, Laurent Maillet, +15
Nature Communications (14 January, 2020)
Urine-derived cells provide a readily accessible cell type for feeder-free mRNA reprogramming
by A. Gaignerie, N. Lefort, M. Rousselle, +13
Scientific Reports (19 September, 2018)