CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq

Single-cell transcriptomics requires a method that is sensitive, accurate, and reproducible. Here, we present CEL-Seq2, a modified version of our CEL-Seq method, with threefold higher sensitivity, lower costs, and less hands-on time. We implemented CEL-Seq2 on Fluidigm’s C1 system, providing its first single-cell, on-chip barcoding method, and we detected gene expression changes accompanying the progression through the cell cycle in mouse fibroblast cells. We also compare with Smart-Seq to demonstrate CEL-Seq2’s increased sensitivity relative to other available methods. Collectively, the improvements make CEL-Seq2 uniquely suited to single-cell RNA-Seq analysis in terms of economics, resolution, and ease of use.Seventh Framework Programme (European Commission)Israel Science Foundatio

CEL-Seq-pipeline: CEL-Seq2 pipeline version 1.0

<p>CEL-Seq2 pipeline version 1.0</p

A Synthetic Oligo Library and Sequencing Approach Reveals an Insulation Mechanism Encoded within Bacterial σ54 Promoters

We use an oligonucleotide library of >10,000 variants to identify an insulation mechanism encoded within a subset of σ54 promoters. Insulation manifests itself as reduced protein expression for a downstream gene that is expressed by transcriptional readthrough. It is strongly associated with the presence of short CT-rich motifs (3–5 bp), positioned within 25 bp upstream of the Shine-Dalgarno (SD) motif of the silenced gene. We provide evidence that insulation is triggered by binding of the ribosome binding site (RBS) to the upstream CT-rich motif. We also show that, in E. coli, insulator sequences are preferentially encoded within σ54 promoters, suggesting an important regulatory role for these sequences in natural contexts. Our findings imply that sequence-specific regulatory effects that are sparsely encoded by short motifs may not be easily detected by lower throughput studies. Such sequence-specific phenomena can be uncovered with a focused oligo library (OL) design that mitigates sequence-related variance, as exemplified herein

The gene regulatory program of Acrobeloides nanus reveals conservation of phylum-specific expression

The evolution of development has been studied through the lens of gene regulation by examining either closely related species or extremely distant animals of different phyla. In nematodes, detailed cell-and stage-specific expression analyses are focused on the model Caenorhabditis elegans, in part leading to the view that the developmental expression of gene cascades in this species is archetypic for the phylum. Here, we compared two species of an intermediate evolutionary distance: the nematodes C. elegans (clade V) and Acrobeloides nanus (clade IV). To examine A. nanus molecularly, we sequenced its genome and identified the expression profiles of all genes throughout embryogenesis. In comparison with C. elegans, A. nanus exhibits a much slower embryonic development and has a capacity for regulative compensation of missing early cells. We detected conserved stages between these species at the transcriptome level, as well as a prominent middevelopmental transition, at which point the two species converge in terms of their gene expression. Interestingly, we found that genes originating at the dawn of the Ecdysozoa super-group show the least expression divergence between these two species. This led us to detect a correlation between the time of expression of a gene and its phylogenetic age: evolutionarily ancient and young genes are enriched for expression in early and late embryogenesis, respectively, whereas Ecdysozoa-specific genes are enriched for expression during the middevelopmental transition. Our results characterize the developmental constraints operating on each individual embryo in terms of developmental stages and genetic evolutionary history

Additional file 6: of CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq

Supplementary file 2. CEL-Seq in C1. The file includes the complete information for performing CEL-Seq on the Fluidigm C1 instrument. (DOCX 17 kb

Additional file 1: Figure S1. of CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq

Optimization of the CEL-Seq protocol. A Number of genes obtained from ten replicates of 100 pg RNA performed with each type of primer: the original primer, the original primer with the inclusion of UMI, and the shortened UMI primer. B Number of transcripts identified for the two primers containing a UMI. C Estimating the efficiency of CEL-Seq using UMIs and ERCC spike-ins. The efficiency is computed as the y-intercept. D Side-by-side comparison of column clean-up, bead clean-up, and two RTs relative to CEL-Seq with a UMI primer. E Side-by-side comparison of different second-strand synthesis enzymes. The MessageAmp II enzyme was the one used originally. (PDF 519 kb

Author correction: The mid-developmental transition and the evolution of animal body plans (Nature, (2016), 531, 7596, (637-641), 10.1038/nature16994)

In this Letter, affiliation number 1 was incorrectly listed as ‘Department of Biology, Technion – Israel Institute of Technion, Haifa 32000, Israel’ instead of ‘Department of Biology, Technion- Israel Institute of Technology, Haifa 32000, Israel’. The original Letter has not been corrected