Systematic optimization of sonication conditions.

<p><b>Part 1. (A)</b> Schematic of the Bioruptor XL water bath with tube positions numbered from 1 to 12 in the left (L) and right (R) carousels; red arrows indicate the alignment marks for carousel assembly and positioning. <b>(B)</b> The effect of sample volume on sonication efficiency at low power setting. Cell suspensions of variable volume (100–700 μL, as indicated) were loaded into positions L3, L4, L5, L9, L10 and L11. Vacant L-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 8 min in 1:4 ELB:H₂O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Remaining intact cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. <i>p</i>-value for analysis of variance between sonicated samples is 1.0×10⁻⁵, between all samples 1.4×10⁻⁶. <i>p</i>-values for selected T-tests are shown on the graph. <b>(C)</b> Reproducibility of sample sonication across positions L1–L12. Sonication was carried out for 40 min in 1:4 ELB:H₂O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Samples were reverse cross-linked overnight and the resulting DNA was purified using the Qiagen PCR clean-up kit followed by 1.1% agarose gel analysis. <b>(D)</b> The effect of sample position and power setting on sonication efficiency. 500 μL cell suspensions were loaded into positions R10-R4 and vacant R-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 1 min in 1:4 ELB:H₂O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation, no ice. Intact remaining cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. <i>p</i>-value for analysis of variance between sonicated samples is 3.6×10⁻⁹ (L-power) and 8.2×10⁻¹⁰ (H-power). <i>p</i>-values for selected T-tests are shown on the graph.</p

Comparison of chromatin fragmentation by ultrasound alone or in combination with benzonase digestion.

<p><b>(A)</b> Comparison of sonication efficiency at the L and H power outputs over time. 500 μL cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for various times (as indicated) in 1:4 ELB:H₂O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Following sonication samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The “Quick” lanes contain samples sonicated for 20 min and reverse cross-linked for 1 h at +60°C. <b>(B)</b> Coomassie Blue staining of protein fractions generated during the sonication time course shown in panel A. Note the absence of high molecular weight proteins after 20 min of ultrasound treatment. <b>(C)</b> Titration of benzonase (0.2U to 90U) to fragment chromatin solubilized by 2 min L-power sonication. Following the digest, samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148023#sec002" target="_blank">Material and Methods</a> section for detailed reaction conditions. <b>(D)</b> Combination of brief sonication (2 min at L-power) and benzonase digestion (0.7U to 90U) preserves the integrity of large proteins.</p

Microsatellite Data for 246 Lasiurus cinereus (14 loci)

Microsatellite genotype scores for 246 hoary bats (Lasiurus cincereus) at 14 loci

Appendix A. A table of 14C dates from Chatsworth Bog and Nelson Lake.

A table of 14C dates from Chatsworth Bog and Nelson Lake

Supplement 1. Data showing percentage C4 grass for samples from Lake Challa and Lake Rutundu.

<h2>File List</h2><div> <p><a href="Supp1.csv">Supp1.csv</a> (MD5: f047c813f5a57757cf0888997686c691)</p> </div><h2>Description</h2><div> <p>Data on the core depth (cm), age (cal. yr BP), number of samples that exceeded the 2σ threshold of blank CO₂ yields, and percentage C₄ grass (with 95% confidence intervals) determined from grass-pollen δ¹³C analyses for each of the samples analyzed from Lake Challa and Lake Rutundu.</p> </div

Hair hydrogen isotope data for 144 Lasiurus borealis

Stable hydrogen isotope ratios of hair for red bats (Lasiurus borealis

Appendix A. Histograms of δ13C values of individual grains of grass pollen for samples from Lake Challa and Lake Rutundu.

Histograms of δ13C values of individual grains of grass pollen for samples from Lake Challa and Lake Rutundu

Genotype scores for 142 Lasiurus borealis (14 loci)

Microsatellite genotype scores for 142 easter red bats (Lasiurus cincereus) at 14 loci