Demand driven approach of DASCOH in Bangladesh

Demand driven approach of DASCOH in Banglades

Differences in gene expression of AdMSCs and FBs.

<p>A) ANOVA with FDR of 1% between undifferentiated AdMSCs (5 replicates) and FBs (6 replicates) recovered 62 differentially expressed genes, 24 with higher and 38 with lower expression in AdMSCs than FBs. The scale shows the up (light red) or down regulation (light blue) in standard deviations from the mean expression for each gene. B) Comparison of differentially expressed genes between AdMSCs and FBs in the undifferentiated state (light grey) and in AdMSC- and FB-derived adipocytes, osteoblasts and chondrocytes using Venn diagram. Many genes remain (light blue) and many differentiation-related genes become (yellow, pink or blue) differentially expressed in AdMSC- and FB-derived differentiated cells. Abbreviations: AdMSC, adipose-derived mesenchymal stem cell; FB, fibroblast; Undif., undifferentiated; Adipo, adipocyte; Osteo, osteoblast; Chondro, chondrocyte; deriv., derived.</p

PCA of cell type-specific gene expression.

<p>9000 most highly expressed genes were analyzed by multi-group ANOVA to find differentially expressed genes between cell types: undifferentiated AdMSCs and FBs, and AdMSC- and FB-derived adipocytes, osteoblasts and chondrocytes using false discovery rate (FDR) of 0.1%. PCA of the resulting 792 genes was used to visualize the relationship of the samples based on annotations such as A) cell type, B) cell origin (AdMSC or FB), C) time groups of differentiation and D) patient. Each circle represents one sample, and is connected by edges to four other most closely related samples in A and B. The same genes that separate different cell types, also separate undifferentiated AdMSCs and FBs and are regulated over time with no differences between patients. However, AdMSCs and FBs retain characteristic gene expression even in the differentiated state. Abbreviations: Undif., undifferentiated; Ad, adipocyte; Os, osteoblast; Ch, chondrocyte; AdMSC, adipose-derived mesenchymal stem cell; FB, fibroblast; P, patient.</p

Distinctly expressed genes between undifferentiated AdMSCs and FBs (based on ANOVA with FDR of 1%).

<p>Distinctly expressed genes between undifferentiated AdMSCs and FBs (based on ANOVA with FDR of 1%).</p

| Optimal conditions for template switching.

<p>The qPCR-derived Ct values for the investigated conditions are shown. Please note that the y-axis scales are broken and that the graphs have different Ct value scales. Also, no error bars are shown for the no template controls (NTC) as these reactions were performed in only one tube. In contrast, the actual experiments were performed in triplicate with the standard deviation error bars displayed in the graphs. The central part of the figure illustrates the template-switching process in the setting of our STRT method. (a) The optimal TSO concentration was 1 μM and (b) shorter TSOs showed a tendency for better performance (see also Table <b>S2</b> and Figure <b>S1</b> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085270#pone.0085270.s001" target="_blank">File S1</a>). The green datapoint in the TSO length graph represents the published version of the TSO [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085270#B10" target="_blank">10</a>] that is 40 bases in length. (c) shows analysis of different RT enzymes. SSII was a better choice than SSIII and, a cycled SSIII protocol (8 cycles of 50°C for 5 min and 60°C for 1 min; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085270#B19" target="_blank">19</a>]) did not work, possibly due to the elevated temperature periods inactivating the enzyme (see also Table <b>S2</b> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085270#pone.0085270.s001" target="_blank">File S1</a>). (b) The optimal SSII amount was 10 units per 10 μl reaction.</p

Base Preferences in Non-Templated Nucleotide Incorporation by MMLV-Derived Reverse Transcriptases

<div><p>Reverse transcriptases derived from Moloney Murine Leukemia Virus (MMLV) have an intrinsic terminal transferase activity, which causes the addition of a few non-templated nucleotides at the 3´ end of cDNA, with a preference for cytosine. This mechanism can be exploited to make the reverse transcriptase switch template from the RNA molecule to a secondary oligonucleotide during first-strand cDNA synthesis, and thereby to introduce arbitrary barcode or adaptor sequences in the cDNA. Because the mechanism is relatively efficient and occurs in a single reaction, it has recently found use in several protocols for single-cell RNA sequencing. However, the base preference of the terminal transferase activity is not known in detail, which may lead to inefficiencies in template switching when starting from tiny amounts of mRNA. Here, we used fully degenerate oligos to determine the exact base preference at the template switching site up to a distance of ten nucleotides. We found a strong preference for guanosine at the first non-templated nucleotide, with a greatly reduced bias at progressively more distant positions. Based on this result, and a number of careful optimizations, we report conditions for efficient template switching for cDNA amplification from single cells.</p> </div

| UMI length.

<p>(a) shows the UMI principle and the output depending on reaction efficiency. If three out of the ten transcript molecules in total are labeled with the unique identifier (i.e. barcoded), only three barcodes will be observed in the sequencing data, translating into a transcript count of three for this particular transcript. Converting eight out of the ten molecules leads to the identification of eight barcodes in the sequencing reads. A UMI can become saturated if the number of transcripts copies exceeds the number of possible UMI combinations.(b) shows the distribution of barcodes for RPLP1 and spike MC28 in the performed reactions. Corresponding graphs for the other investigated transcripts are shown in Figure <b>S6</b> and Figure <b>S7</b> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085270#pone.0085270.s001" target="_blank">File S1</a> for the analyzed RNA spikes. The reactions with the N10rG3 TSOs exhibited the highest complexity, followed by the reactions employing the N12rG3 and, lastly, the N10rN3 oligonucleotides. In (c) 6-base and 4-base barcodes were extracted from the MALAT1, RPLP1, MT2A, AHSG and CNIH4 reads. The red numbers indicate that the UMI has become saturated.</p

Cell differentiation.

<p>A) AdMSCs and FBs were isolated from two patients (P1, P2) and differentiated towards adipocytes, osteoblasts and chondrocytes. RNA was isolated on days 0–7 during differentiation and the resulting 96 RNA samples were used to generate single sequencing library for gene expression analysis. B) <i>In vitro</i> differentiation of AdMSCs (upper panel) and FBs (lower panel) was confirmed by ORO staining of adipocyte, ARS staining of osteoblast and AB staining of chondrocyte cultures on day 14 upon induction of differentiation. The quantified stainings of FBs are represented relative to AdMSCs (lower panel; AdMSC = 1). Abbreviations: P, patient; AdMSC, adipose-derived mesenchymal stem cell; FB, fibroblast; Ad, adipocyte; Os, osteoblast; Ch, chondrocyte; d, day; ORO, Oil Red O; ARS, Alizarin Red S; AB, Alcian Blue.</p

Principal component analysis (PCA) of non-filtered data.

<p>9000 most highly expressed genes were visualized by PCA based on cell type (undifferentiated, adipocytes, osteoblasts and chondrocytes) without prior statistical filtering. Different cell types cluster together upon PCA. Abbreviations: Undif., undifferentiated; Ad, adipocyte; Os, osteoblast; Ch, chondrocyte.</p

The list of genes that are highly expressed in AdMSCs and chondrocytes but not in FBs.

<p>The list of genes that are highly expressed in AdMSCs and chondrocytes but not in FBs.</p