scDual-Seq: mapping the gene regulatory program of Salmonella infection by host and pathogen single-cell RNA-sequencing

The interaction between a pathogen and a host is a highly dynamic process in which both agents activate complex programs. Here, we introduce a single-cell RNA-sequencing method, scDual-Seq, that simultaneously captures both host and pathogen transcriptomes. We use it to study the process of infection of individual mouse macrophages with the intracellular pathogen Salmonella typhimurium. Among the infected macrophages, we find three subpopulations and we show evidence for a linear progression through these subpopulations, supporting a model in which these three states correspond to consecutive stages of infection. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1340-x) contains supplementary material, which is available to authorized users

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

Core promoter T-blocks correlate with gene expression levels in C. elegans

Core promoters mediate transcription initiation by the integration of diverse regulatory signals encoded in the proximal promoter and enhancers. It has been suggested that genes under simple regulation may have low-complexity permissive promoters. For these genes, the core promoter may serve as the principal regulatory element; however, the mechanism by which this occurs is unclear. We report here a periodic poly-thymine motif, which we term T-blocks, enriched in occurrences within core promoter forward strands in Caenorhabditis elegans. An increasing number of T-blocks on either strand is associated with increasing nucleosome eviction. Strikingly, only forward strand T-blocks are correlated with expression levels, whereby genes with ≥6 T-blocks have fivefold higher expression levels than genes with ≤3 T-blocks. We further demonstrate that differences in T-block numbers between strains predictably affect expression levels of orthologs. Highly expressed genes and genes in operons tend to have a large number of T-blocks, as well as the previously characterized SL1 motif involved in trans-splicing. The presence of T-blocks thus correlates with low nucleosome occupancy and the precision of a trans-splicing motif, suggesting its role at both the DNA and RNA levels. Collectively, our results suggest that core promoters may tune gene expression levels through the occurrences of T-blocks, independently of the spatio-temporal regulation mediated by the proximal promoter

CEL-Seq: Single-Cell RNA-Seq by Multiplexed Linear Amplification

High-throughput sequencing has allowed for unprecedented detail in gene expression analyses, yet its efficient application to single cells is challenged by the small starting amounts of RNA. We have developed CEL-Seq, a method for overcoming this limitation by barcoding and pooling samples before linearly amplifying mRNA with the use of one round of in vitro transcription. We show that CEL-Seq gives more reproducible, linear, and sensitive results than a PCR-based amplification method. We demonstrate the power of this method by studying early C. elegans embryonic development at single-cell resolution. Differential distribution of transcripts between sister cells is seen as early as the two-cell stage embryo, and zygotic expression in the somatic cell lineages is enriched for transcription factors. The robust transcriptome quantifications enabled by CEL-Seq will be useful for transcriptomic analyses of complex tissues containing populations of diverse cell types

The Roles of the Catalytic and Noncatalytic Activities of Rpd3L and Rpd3S in the Regulation of Gene Transcription in Yeast

<div><p>In budding yeasts, the histone deacetylase Rpd3 resides in two different complexes called Rpd3L (large) and Rpd3S (small) that exert opposing effects on the transcription of meiosis-specific genes. By introducing mutations that disrupt the integrity and function of either Rpd3L or Rpd3S, we show here that Rpd3 function is determined by its association with either of these complexes. Specifically, the catalytic activity of Rpd3S activates the transcription of the two major positive regulators of meiosis, <i>IME1</i> and <i>IME2</i>, under all growth conditions and activates the transcription of <i>NDT80</i> only during vegetative growth. In contrast, the effects of Rpd3L depends on nutrients; it represses or activates transcription in the presence or absence of a nitrogen source, respectively. Further, we show that transcriptional activation does not correlate with histone H4 deacetylation, suggesting an effect on a nonhistone protein. Comparison of <i>rpd3</i>-null and catalytic-site point mutants revealed an inhibitory activity that is independent of either the catalytic activity of Rpd3 or the integrity of Rpd3L and Rpd3S. </p> </div

An upstream repressor element plays a role in Igf2 imprinting

The imprinted Igf2 gene is associated with a small upstream region that is differentially methylated on the active paternal allele. We have identified a repressor element within this sequence and shown that repression is probably mediated through a trans- acting factor, GCF2. DNA methylation of this site abrogates both protein binding and repressor activity. Targeting experiments demonstrate that this element plays a role in the repression of the maternal Igf2 gene in vivo

Additional file 1: of scDual-Seq: mapping the gene regulatory program of Salmonella infection by host and pathogen single-cell RNA-sequencing

scDual-Seq protocol. This file includes the detailed scDual-Seq protocol. (PDF 265 kb

Repression of transcription by Rpd3 requires Rpd3L and Rpd3S.

<p>Cells carrying the <i>UAS</i>_<i>GAL1</i><i>-</i>UAS_<i>HIS4</i><i>-his4-lacZ</i> reporter gene were grown in SD medium to a density of 10⁷ cells/ml. The activity of β-galactosidase (Miller units) in cells expressing Gal4(dbd)-Rpd3 is relative to the level in the control cells (c) expressing only Gal4(dbd). Diploid strains used were as follows: wild-type (Y1884), <i>rco1</i>Δ/<i>rco1</i>Δ (Y1814), <i>sds3</i>Δ/<i>sds3</i>Δ (Y1845), <i>dep1</i>Δ/<i>dep1</i>Δ (Y1894), <i>ume6</i>Δ/<i>ume6</i>Δ (Y1388), and <i>rco1</i>Δ/<i>rco1</i>Δ <i>sds3</i>Δ/<i>sds3</i>Δ (Y1883). These strains carried either <i>pADH1-gal4</i>(dbd)<i>-</i>RPD3 (YEp2593) or <i>pADH1-gal4</i>(dbd) (YEp2149) on a 2-µ vector. Proteins were extracted from at least three independent transformants. </p

Possible molecular mechanisms of transcriptional activation by Rpd3.

<p>A. Deletion of <i>SUM1</i> did not suppress <i>rpd3</i>Δ. Isogenic wild-type (Y1631, closed black squares), <i>rpd3</i>Δ/<i>rpd3</i>Δ (Y1888, empty black squares, dashed line) and <i>rpd3</i>Δ/<i>rpd3</i>Δ <i>sum1</i>Δ<i>/sum1</i>Δ (Y1914, gray circles, dashed lines). B. Ectopic transcription of <i>NDT80</i> partially suppressed the effect of <i>rpd3</i>Δ on the transcription of <i>SPS1</i> and nuclear division. Isogenic <i>NDT80</i>/<i>NDT80</i> (Y1631, black squares), <i>IME2p-6xHA-NDT80/IME2p-6xHA-NDT80</i> (Y1763, gray triangles), <i>rpd3</i>Δ/<i>rpd3</i>Δ (Y1537, empty gray squares, dashed gray lines) and <i>rpd3</i>Δ/<i>rpd3</i>Δ <i>IME2p-6xHA-NDT80/IME2p-6xHA-NDT80</i> (Y1870, empty gray triangle, dashed gray lines) cells were shifted to meiotic conditions (SPM medium), and at the indicated times, samples were taken for RNA extraction and DAPI staining to determine the percentage of cells with more than 1 nucleus. <i>NDT80</i> expression was measured using q-RT PCR. Levels of expression are relative to that of <i>ACT1</i>. The results of a representative experiment are shown. Similar results were obtained from three independent experiments.</p

Diploids with <i>DEP1/RCO1</i> or <i>SDS3/RCO1</i> deletions arrest in meiosis before nuclear division.

<p>Isogenic wt (Y1631, closed squares), <i>dep1∆/dep1∆</i> (Y1894, open circles), and <i>rco1∆/rco1∆ </i><i>sds3∆/sds3∆</i> (Y1883, open triangle) diploids were shifted to meiotic conditions (SPM medium). Samples were taken at the indicated times for FACS analysis to calculate the percentage of cells with 4C DNA content and to count the percentage of cells with more than 2 nuclei (DAPI stain).</p