Estimates of FLEXI RNA abundance using a linear regression model and droplet digital PCR.

<p>(A) Linear regression models (<i>lrm</i>) of molecule per cell values based on RPM values in TGIRT-seq datasets of sncRNAs with literature reported copy number per cell values. The scatter plots show the relationship between log<sub>10</sub>-transformed copy number per cell values reported in the literature [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1011416#pgen.1011416.ref033" target="_blank">33</a>] and log<sub>10</sub>-transformed RPM of sncRNAs in combined TGIRT-seq datasets for each of the 4 cellular RNA samples. The linear regression was plotted as a light blue line with 95% confidence intervals shown as dashed blue lines. sncRNAs used in the linear regression were major spliceosomal snRNAs U1, U2, U4, U5, and U6; minor spliceosomal snRNAs U4ATAC and U6ATAC; U7, 7SL and 7SK RNA; MRP and RNase P RNA (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1011416#pgen.1011416.s025" target="_blank">S4 Table</a>)[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1011416#pgen.1011416.ref033" target="_blank">33</a>]. Pearson (<i>r</i>) correlation coefficients are shown in the upper left of each panel. The Table below the plots shows literature values for three sncRNAs used as standards for droplet digital PCR (ddPCR) and copy number per cell values for these RNAs in different cellular RNA samples based on the <i>lrm</i>. (B) Comparison of abundance estimates for FLEXI RNAs based on the TGIRT-seq <i>lrm</i> and droplet digital PCR (ddPCR). The left-hand columns (TGIRT-seq) show RPM and copy number per cell values of FLEXIs estimated using the TGIRT-seq <i>lrm</i> for the cellular RNA samples. The right-hand columns (ddPCR) show copy number per cell values based on ddPCR abundance relative to 3 different sncRNA standards run in parallel (U7, SNORD14B, and SNORD44). For ddPCR, reverse transcription was performed on DNase-treated, size-selected (≤200 nt) RNA preparations using Maxima H reverse transcriptase with a gene-specific primer at the 3’ end of the putative FLEXI or sncRNA (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1011416#pgen.1011416.s030" target="_blank">S9 Table</a>). ddPCR was then performed using primer sets within the FLEXI sequence or spanning the 5’-exon-FLEXI junction, with the latter subtracted from the FLEXI signal when applicable (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1011416#pgen.1011416.s030" target="_blank">S9 Table</a>). Copy number per cell values for FLEXIs were calculated based on copy number per cell values for U7, SNORD14B, and SNORD44 in each cellular RNA sample obtained using the <i>lrm</i> (Table at the bottom of panel A).</p> <p>(PDF)</p>