Comparing throughput and scalability between single cell RNA-Seq methods.

<p>The reaction volumes, time and cost per cell are estimated for Hi-SCL RNA-Seq and compared to the CEL-Seq method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116328#pone.0116328.ref007" target="_blank">7</a>] and the Fluidigm C1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116328#pone.0116328.ref013" target="_blank">13</a>] as reported in the literature. Sequencing was not included in the estimations. A breakup of the Hi-SCL protocol is provided in Table A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116328#pone.0116328.s001" target="_blank">S1 File</a>.</p><p>Comparing throughput and scalability between single cell RNA-Seq methods.</p

Exclusivity of barcodes and classifying cells by expression.

<p><b>A)</b> The number of unique RNA transcripts per barcode is plotted for barcodes with more than 200 transcripts coming from a mixed sample of 75% mouse and 25% human cells. Transcripts are included if they map either exclusively to the mouse genome (red) or the human genome (blue). <b>B)</b> The distribution of the number of human transcripts found in barcodes of murine cells (top), the number of murine transcripts found in human cells (middle) and the number of total foreign transcripts expected per barcode calculated as the distribution of the sum of pairs of numbers randomly sampled from the two distributions above. <b>C)</b> After clustering 451 murine cells according to their transcriptomes, the correlation of all murine cell transcriptomes with the aggregated transcriptome of cluster #1 is plotted vs. their correlation with aggregated transcriptome of cluster #2. Green cells are mES, comprising 99% of cells in cluster #1, which consists of 290 cells while mEF cells are red, comprising 86% of cells in cluster #2, which consists of 161 cells. <b>D)</b> The same clustering method was repeated after replacing some of the transcripts in each cell with foreign transcripts randomly chosen from the aggregated transcriptome of all cells. The fraction of correctly classified cells averaged over 20 clustering trials is plotted vs. the number of foreign transcripts that were introduced in each cell before clustering.</p

Microfluidic chip design.

<p><b>A)</b> 96 parallel drop-makers sharing the same oil inlet and the same collection outlet are used as one chip to encapsulate the barcode library emulsion. The device is multilayered, with 325 μm thick distribution channels (black) and 25 μm thick drop makers (gray). <b>B)</b> Co-flow drop maker with separate inlets for cells and lysing buffer that mix at encapsulation. <b>C)</b> “Three point merger” device re-injecting two emulsions and adding buffer at the coalescence junction. The electrode channels are filled with solder to create the electrodes that mediated coalescence at the junction.</p

H2A ubiquitination activity of Ring1B is essential for the maintenance of ESC identity and repression of target gene expression.

<p>(A) Morphology of OHT-untreated and –treated (day 8) <i>Ring1A^−/−; Ring1B^fl/fl; R26::CreERT2</i> ESC lines expressing the indicated transgene. The images were acquired under a phase-contrast microscope. Scale bars indicate 200 µm. (B) Histograms showing the expression changes of H2AK119u1+ and H2AK119u1− genes in <i>Ring1A^−/−; Ring1B^fl/fl; Rosa26::CreERT2</i> ESCs expressing mock (blue line), WT Ring1B (red line), or mutant Ring1B (green dotted line) following OHT treatment. (C) Expression levels of <i>Hoxa9</i>, <i>Hoxb13</i>, <i>Hoxd11</i>, <i>Zic1</i>, <i>Pax3</i> and <i>Pou5f1</i> in <i>Ring1A^−/−; Ring1B^fl/fl; Rosa26::CreERT2</i> ESCs expressing mock, WT, I53S, or I53A Ring1B before (−) or after (+) OHT treatment (day 2). Expression levels were normalized to a <i>Gapdh</i> control and are depicted as fold changes relative to mock (OHT-untreated) ESCs. Error bars represent standard deviation determined from at least three independent experiments. (D) Local levels of trimethylated H3K4 (H3K4me3) at promoter regions of representative target genes in <i>Ring1A^−/−; Ring1B^fl/fl; R26::CreERT2</i> ESCs stably expressing mock, WT, I53S, or I53A Ring1B before (−) or after (+) OHT treatment (day 2) were determined by ChIP and site-specific real-time PCR. The relative amount of immunoprecipitated DNA is depicted as a percentage of input DNA. Error bars represent standard deviation determined from at least three independent experiments. (E) As in (D), but showing local levels of RNA polymerase II (RNAP) detected with the 8WG16 antibody.</p

Global mapping of Ring1B-dependent H2AK119u1 deposition in ESCs reveals that genes occupied by H2AK119u1 represent central targets of PRC1.

<p>(A) ChIP-on-chip analysis showing the average of H2AK119u1 distributions at the promoter regions (from −5 kb to +5 kb relative to TSS) of Ring1B-bound and –unbound genes in <i>Ring1A^−/−</i> (OHT−: green line) and <i>Ring1A/B</i>-dKO (OHT+: red line) ESCs. Enrichment of H2AK119u1 (obtained by E6C5 mAb) and H2A is expressed relative to input DNA, and H2AK119u1 is normalized to H2A. (B) Venn diagram representing the overlap among genes occupied by Ring1B, H2AK119u1 and H3K27me3. Numbers in parentheses represent the total number of genes occupied by each one. (C) Graphic representation of expression changes induced by <i>Ring1B</i> depletion (2 days after OHT treatment) for each subset of genes classified by the presence (+) or absence (−) of Ring1B, H2AK119u1 and H3K27me3 is shown. The average, deviation and distribution of the expression changes for the respective subsets of genes determined by microarray analysis are shown. The 95% Confidence interval (CI) and standard deviation (SD) for the average value of the expression change are indicated. Significant (<i>P</i><0.001) and insignificant (<i>P</i>≥0.01) expression changes were determined by the Student's <i>t</i>-test and are indicated in orange and grey, respectively. <i>P</i>-values for the difference of expression changes between the indicated 2 groups are calculated by the Student's <i>t</i>-test and are indicated above each graph.</p

Generation of ESCs expressing catalytically inactive Ring1B.

<p>(A) Schematic representation of 3xFlag-tagged Ring1B, showing wild-type and point-mutant derivatives. Each of these construct was stably transfected into <i>Ring1A^−/−; Ring1B^fl/fl; R26::CreERT2</i> ESCs. (B) Immunoblot analysis of Ring1A, Ring1B, Flag, H2AK119u1 and Lamin B protein levels in whole cell lysates of <i>wild-type</i> and <i>Ring1A^−/−; Ring1B^fl/fl; R26::CreERT2</i> ESC lines expressing mock, WT, I53S, or I53A Ring1B with or without OHT treatment (OHT+ and −, respectively). (C) Immunoprecipitation (IP) analysis showing the association of exogenous Ring1B WT, I53S or I53A with an endogenous PRC1 component Mel18. Extracts of OHT-untreated (−) and -treated [(+); day 2] <i>Ring1A^−/−; Ring1B^fl/fl; R26::CreERT2</i> ESC lines expressing each of the constructs were immunoprecipitated with anti-Flag antibody. Resulting precipitates (IP) and lysates (Input) were immunoblotted with antibodies against Flag, Ring1B and Mel18. (D) Association of Flag-tagged proteins in <i>Ring1A^−/−; Ring1B^fl/fl; R26::CreERT2</i> ESC lines stably expressing mock, Flag-tagged Ring1B WT, I53S, or I53A with promoter regions of their representative target genes before (−) or after (+) OHT treatment (day 2) as determined by ChIP and site-specific real-time PCR. Error bars represent standard deviations determined from three independent experiments. (E) 3D FISH with probe pairs at <i>Hoxb</i> locus (<i>Hoxb1</i> and <i>Hoxb13</i>) in PFA-fixed nuclei of <i>Ring1A^−/−; Ring1B^fl/fl; R26::CreERT2</i> ESC lines stably expressing mock, WT, I53S, or I53A Ring1B before (−) or after (+) OHT treatment (day 2). Scale bars indicate 1 µm. The boxes show the median and interquartile range of interprobe distances (µm) in the indicated cells. Open circles indicate outliers. The statistical significance of differences between the indicated two data was examined by the Mann-Whitney U test.</p

A schematic summary of this study demonstrating that PRC1-dependent repression of developmental genes in ES cells is mediated by multiple effector mechanisms.

<p>A schematic summary of this study demonstrating that PRC1-dependent repression of developmental genes in ES cells is mediated by multiple effector mechanisms.</p