diffRepeats: Quantify repeat element enrichment with next-generation sequencing data

<p>Quantify repeat element enrichment with next-generation sequencing data</p

Effect of <i>GGT</i> insertion on the expression of a downstream <i>gfp</i> reporter in G418-resistant cell lines.

a<p>difference from GGT, p<0.0003, paired t-test.</p>b<p>difference from GGT, p<0.0003, paired t-test.</p

Read-through activation in Neo-Luc cell lines.

<p>Total RNA prepared from individual cell lines isolated after electroporation with 1 µg of DNA was assayed by hybridization and protection from nuclease S1 digestion. Expression of <i>E1a-neo,</i> read-through transcription (RT), and <i>E1b-luc</i> produced the specific protected bands indicated in the diagram below the autoradiographic data. The diagram is laid out as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015704#pone-0015704-g004" target="_blank">Fig. 4</a>. Each lane represents a different cell line. He: Hela cells; DPM: cell lines derived from pNeoDPME1bLuc; GGT: cell lines derived from pNeoGGTE1bLuc; M: size standards.</p

Read-through activation in Neo-GFP cell lines.

<p>A. Nuclear RNA prepared from pooled cultures of cell lines isolated after electroporation with 1 µg of DNA was assayed by hybridization and protection from nuclease S1 digestion. Expression of <i>E1a-neo,</i> read-through transcription (RT), and <i>E1b-gfp</i> produced the specific protected bands indicated in the diagram below the autoradiographic data. The arrows indicate the relative positions of the transcripts in the template (the uncertain end of the read-through transcript is indicated by the dashed line). The probe is indicated by a line with the position of the 5′-end label shown as an asterisk. The region of the probe protected by each transcript is indicated as double-stranded (DNA-RNA hybrid). The position of divergence of the sequence of the probe for read-through transcription from the read-through RNA product is shown by the loss of DNA-RNA hybrid formation. Variation in migration of the RT and E1a-GFP products probably was caused by sequence differences at the junction site produced during plasmid construction. Lane designations: 1: HeLa cells; 2: culture derived from pNeoE1bGFP; 3: culture derived from pNeoGGTE1bGFP; 4: culture derived from pNeoDPME1bGFP. The positions of size markers (not shown) are indicated on the left of the autoradiograms. B. <i>E1b-GFP</i> RNA levels from two experiments (Expt 1 is shown in A) were quantified and normalized to the quantity of <i>E1a-Neo</i> RNA. The results are expressed relative to the pNeoE1bGFP value (1.00).</p

Histograms of GFP-expression in cells from pooled colonies analyzed by flow cytometry.

<p>The plots from Experiment 2 of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015704#pone-0015704-t002" target="_blank">Table 2</a> show the gating for the threshold of GFP-positive designation. The plasmid used to generate the colonies is indicated above each histogram.</p

Effect of GGT insertion on GFP expression in pooled cultures of G418-resistant colonies^a.

a<p>Colonies (collected from three plates per plasmid) in pooled cultures were harvested and analyzed by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015704#s4" target="_blank">Materials and Methods</a>.</p>b<p>percentage of cells in the population with FL1-H>10^1.1.</p>c<p>per cell.</p>d<p>not done.</p

Genetic system for analysis of read-through activation.

<p>A. Organization of the region of the adenovirus 5 genome to the left of the E1b transcription start. The top diagram shows the relationship of the two major transcription initiation sites (E1a, nucleotide position 499; and E1b, 1702) and the position of insertion of the ectopic translation termination sequence <i>GGT</i> (1339). The E1a enhancer region is indicated as a filled rectangle and alternate E1a transcription initiation sites as arrows. The E1a coding exons are shaded (exon 1) and open (exon 2) rectangles. Below is an expanded view of the E1b transcriptional control region indicating mapped sites of DNA-protein interaction in the distal region (I-V), and proximal binding sites for Sp1 (GC) and TBP (AT). The locations of the E1a translation termination codon and poly(A) addition site within the E1b control region also are indicated. B. Selection marker-reporter plasmids: structures of plasmids with <i>neo</i> and <i>gfp</i> or <i>luc</i> gene replacements. pE1a: E1a promoter; Neo: G418 resistance gene; T: termination sequences, <i>GGT</i> or <i>DPM</i>; pE1b: E1b promoter; GFP: green fluorescent protein gene; Luc: luciferase gene.</p

Integrated NeoGGTE1bGFP sequences in stable cell lines.

<p>DNA was extracted from G418-resistant stable cell lines established with the plasmid pNeoGGTE1bGFP (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015704#pone-0015704-g001" target="_blank">Fig. 1b</a>). Blots were prepared and probed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015704#s4" target="_blank">Materials and Methods</a>. Lane designations: M, size markers derived from an <i>Eco</i>RI/<i>Psh</i>A1 digest of the plasmid (sizes shown on the left); 2, reconstruction with 2 copies of <i>Sph</i>I-digested (linear) plasmid (7.2 kbp), based on quasi-tetraploid human DNA content; 20, reconstruction with 20 copies of plasmid; A+, GFP-expressing cell line 1 isolated after electroporation of 1 µg of DNA; A−, GFP-negative cell line from the same experiment as A+; B+, GFP-expressing cell line isolated in a second experiment after electroporation of 1 µg of DNA; B−, GFP-negative cell line from the same experiment as B+; C+, GFP-expressing cell line isolated after lipofection with 1 µg of DNA; C−, GFP-negative cell line 2 from the same experiment as C+. An irrelevant lane was removed from between lanes 20 and A+. The diagram below shows the results expected from a single (top) or tandem (bottom) integration of the complete plasmid sequence (open box). For the former, the sizes of the two junction DNA fragments that hybridize to the probe (shaded box) will depend on the site of insertion. For the latter, in addition to the junction fragments, a unit length band (7.2 kbp) will be generated and its intensity will depend on the number of copies in the tandem array. There is a single site for <i>Sph</i>I (S) cleavage in the plasmid <i>neo</i> gene.</p

Effect of read-through transcription on E1b-Luc RNA expression in stable cell lines^a.

a<p>Monolayers at 70-90% confluence were harvested and the RNAs indicated were assayed and quantified from the experiment shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015704#pone-0015704-g004" target="_blank">Fig. 4</a>. All units are arbitrary.</p>b<p>E1a-Luc/E1a-Neo; mean +/− st. dev: GGT cell lines: 0.90+/−0.20; DPM cell lines: 1.42+/−0.25; difference between GGT and DPM, p<0.006, unpaired t-test.</p>c<p>Normalized to E1a-Neo.</p>d<p>Not determined.</p

Correction to “Effects of Heteroatoms of Tetracene and Pentacene Derivatives on Their Stability and Singlet Fission”

Correction to “Effects of Heteroatoms of Tetracene and Pentacene Derivatives on Their Stability and Singlet Fission