BRB-seq: ultra-affordable high-throughput transcriptomics enabled by bulk RNA barcoding and sequencing

Alpern, Daniel; Gardeux, Vincent; Russeil, Julie; Mangeat, Bastien; Meireles-Filho, Antonio C. A.; Breysse, Romane; Hacker, David L.; Deplancke, Bart

doi:10.1186/s13059-019-1671-x

Cited by 146 publications

(184 citation statements)

References 41 publications

(46 reference statements)

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“…Cell culture RNA-seq libraries were prepared by BRB-seq 59 . For proliferation assays, TRE dCas9-KRAB XCL4 cells were infected in quadruplicate with gRNAs targeting 7 ASD-genes and 1 nontargeting gRNA.…”

Section: Methodsmentioning

confidence: 99%

High-throughput single-cell functional elucidation of neurodevelopmental disease-associated genes reveals convergent mechanisms altering neuronal differentiation

Lalli

Avey

Dougherty

et al. 2019

Preprint

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The overwhelming success of exome-and genome-wide association studies in discovering thousands of disease-associated genes necessitates novel high-throughput functional genomics approaches to elucidate the mechanisms of these genes. Here, we have coupled multiplexed repression of neurodevelopmental diseaseassociated genes to single-cell transcriptional profiling in differentiating human neurons to rapidly assay the functions of multiple genes in a disease-relevant context, assess potentially convergent mechanisms, and prioritize genes for specific functional assays. For a set of 13 autism spectrum disorder (ASD) associated genes, we demonstrate that this approach generated important mechanistic insights, revealing two functionally convergent modules of ASD genes: one that delays neuron differentiation and one that accelerates it. Five genes that delay neuron differentiation (ADNP, ARID1B, ASH1L, CHD2, and DYRK1A) mechanistically converge, as they all dysregulate genes involved in cell-cycle control and progenitor cell proliferation. Live-cell imaging after individual ASD gene repression validated this functional module, confirming that these genes reduce neural progenitor cell proliferation and neurite growth. Finally, these functionally convergent ASD gene modules predicted shared clinical phenotypes among individuals with mutations in these genes. Altogether these results demonstrate the utility of a novel and simple approach for the rapid functional elucidation of neurodevelopmental disease-associated genes.

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Section: Methodsmentioning

confidence: 99%

High-throughput single-cell functional elucidation of neurodevelopmental disease-associated genes reveals convergent mechanisms altering neuronal differentiation

Lalli

Avey

Dougherty

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…Such early pooling (or early multiplexing) of samples reduces the costs per individual sample and increases throughput. Recently, BRB-seq successfully adapted the early multiplexing approach of single-cell protocols to bulk RNA-seq (Alpern et al 2019). However, such feature has two main caveats: first, the identity of the individual samples is only recovered during bioinformatic analysis, and second, any variation in RNA input amount will result in high variation in sequencing coverage per sample (see the SMART-seq2 method for an exception (Picelli et al 2014b)).…”

Section: Rnaseq Mrna Extraction Transcriptomics Tagseq Tm3seqmentioning

confidence: 99%

TM3’seq: A Tagmentation-Mediated 3’ Sequencing Approach for Improving Scalability of RNAseq Experiments

Pallares

Picard

Ayroles

2020

G3 Genes|Genomes|Genetics

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RNA-seq has become the standard tool for collecting genome-wide expression data in diverse fields, from quantitative genetics and medical genomics to ecology and developmental biology. However, RNA-seq library preparation is still prohibitive for many laboratories. Recently, the field of single-cell transcriptomics has reduced costs and increased throughput by adopting early barcoding and pooling of individual samples —producing a single final library containing all samples. In contrast, RNA-seq protocols where each sample is processed individually are significantly more expensive and lower throughput than single-cell approaches. Yet, many projects depend on individual library generation to preserve important samples or for follow-up re-sequencing experiments. Improving on currently available RNA-seq methods we have developed TM3′seq, a 3′-enriched library preparation protocol that uses Tn5 transposase and preserves sample identity at each step. TM3′seq is designed for high-throughput processing of individual samples (96 samples in 6h, with only 3h hands-on time) at a fraction of the cost of commercial kits ($1.5 per sample). The protocol was tested in a range of human and Drosophila melanogaster RNA samples, recovering transcriptomes of the same quality and reliability than the commercial NEBNext kit. We expect that the cost- and time-efficient features of TM3′seq make large-scale RNA-seq experiments more permissive for the entire scientific community.

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“…SCRB-seq [17] was originally developed for single-cell analysis, which adds both cell barcodes and UMIs to cDNAs by early multiplexing, then specifically enriches 3 fragments for gene expression quantification. This method was used in bulk RNA-seq analysis in several studies and then was further optimized to form an approach called BRB-seq [18]. By 3 end barcoding and enrichment, BRB-seq is able to produce dozens of libraries at a very low cost, which showed great potential for DE analysis.…”

Section: Identification Of Differentially Expressed Genes (Degs)mentioning

confidence: 99%

Decode-seq: a practical approach to improve differential gene expression analysis

Yang

HuJun

et al. 2020

Genome Biol

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Many differential gene expression analyses are conducted with an inadequate number of biological replicates. We describe an easy and effective RNA-seq approach using molecular barcoding to enable profiling of a large number of replicates simultaneously. This approach significantly improves the performance of differential gene expression analysis. Using this approach in medaka (Oryzias latipes), we discover novel genes with sexually dimorphic expression and genes necessary for germ cell development. Our results also demonstrate why the common practice of using only three replicates in differential gene expression analysis should be abandoned.

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BRB-seq: ultra-affordable high-throughput transcriptomics enabled by bulk RNA barcoding and sequencing

Cited by 146 publications

References 41 publications

High-throughput single-cell functional elucidation of neurodevelopmental disease-associated genes reveals convergent mechanisms altering neuronal differentiation

High-throughput single-cell functional elucidation of neurodevelopmental disease-associated genes reveals convergent mechanisms altering neuronal differentiation

TM3’seq: A Tagmentation-Mediated 3’ Sequencing Approach for Improving Scalability of RNAseq Experiments

Decode-seq: a practical approach to improve differential gene expression analysis

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