Multiplexed Illumina sequencing libraries from picogram quantities of DNA

Bowman, Sarah K.; Simon, Matthew D.; Deaton, Aimée M.; Tolstorukov, Michael Y.; Borowsky, Mark; Kingston, Robert E.

doi:10.1186/1471-2164-14-466

Cited by 79 publications

(76 citation statements)

References 17 publications

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“…For Sanger sequencing of the cprA gene, the primers used are listed in Table S1 in the supplemental material. For whole-genome sequencing, a random-fragment library was constructed from each genome using a custom protocol, as described previously (34). Briefly, genomic DNA was purified using the MCD 85201 kit (Epicentre Biotechnologies, Madison, WI), and 2 g was sheared using a Covaris S2 sonicator (5% intensity; 10% duty cycle; 200 cycles/burst, processing time, 4 min).…”

Section: Methodsmentioning

confidence: 99%

Pseudomonas aeruginosa High-Level Resistance to Polymyxins and Other Antimicrobial Peptides Requires cprA , a Gene That Is Disrupted in the PAO1 Strain

Gutu

Rodgers

Park

et al. 2015

Antimicrob Agents Chemother

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The arn locus, found in many Gram-negative bacterial pathogens, mediates resistance to polymyxins and other cationic antimicrobial peptides through 4-amino-L-arabinose modification of the lipid A moiety of lipopolysaccharide. In Pseudomonas aeruginosa, several two-component regulatory systems (TCSs) control the arn locus, which is necessary but not sufficient for these resistance phenotypes. A previous transposon mutagenesis screen to identify additional polymyxin resistance genes that these systems regulate implicated an open reading frame designated PA1559 in the genome of the P. aeruginosa PAO1 strain. Resequencing of this chromosomal region and bioinformatics analysis for a variety of P. aeruginosa strains revealed that in the sequenced PAO1 strain, a guanine deletion at the end of PA1559 results in a frameshift and truncation of a full-length open reading frame that also encompasses PA1560 in non-PAO1 strains, such as P. aeruginosa PAK. Deletion analysis in the PAK strain showed that this full-length open reading frame, designated cprA, is necessary for polymyxin resistance conferred by activating mutations in the PhoPQ, PmrAB, and CprRS TCSs. The cprA gene was also required for PmrAB-mediated resistance to other cationic antimicrobial peptides in the PAK strain. Repair of the mutated cprA allele in the PAO1 strain restored polymyxin resistance conferred by an activating TCS mutation. The deletion of cprA did not affect the arn-mediated lipid A modification, indicating that the CprA protein is necessary for a different aspect of polymyxin resistance. This protein has a domain structure with a strong similarity to the extended short-chain dehydrogenase/reductase family that comprises isomerases, lyases, and oxidoreductases. These results suggest a new avenue through which to pursue targeted inhibition of polymyxin resistance. P olymyxins (Pm) are acylated cationic peptides active against Pseudomonas aeruginosa and other Gram-negative pathogens. As pharmaceuticals, Pm B sulfate (PMB) and colistimethate, the prodrug form of colistin (also known as Pm E), are increasingly used to treat multidrug-resistant strains of P. aeruginosa that cause serious infections in critically ill patients and in those with cystic fibrosis (CF) (1, 2). Multidrug-resistant strains emerge when firstline agents, such as antipseudomonal ␤-lactams, aminoglycosides, and fluoroquinolones, are used repeatedly, imposing major selective pressure (3-5). Unfortunately, the prevalence of clinical isolates of P. aeruginosa and other Gram-negative pathogens resistant to PMB and colistin is increasing (6-13).Pm binds to lipopolysaccharide (LPS), the major constituent of the Gram-negative outer membrane, promoting its own uptake and diffusion through the periplasm to the inner membrane, where it disrupts cellular respiration and leads to cell lysis (14). The resistance of Gram-negative pathogens to Pm and other cationic antimicrobial peptides (CAPs) is associated with covalent modification of LPS, namely, the addition of 4-amino-L-arabinose (L-Ara4...

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Section: Methodsmentioning

confidence: 99%

Pseudomonas aeruginosa High-Level Resistance to Polymyxins and Other Antimicrobial Peptides Requires cprA , a Gene That Is Disrupted in the PAO1 Strain

Gutu

Rodgers

Park

et al. 2015

Antimicrob Agents Chemother

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“…Libraries were prepared as previously described (Bowman et al 2013) with modifications outlined in the Supplemental Material.…”

Section: Chip-seqmentioning

confidence: 99%

The E3 ubiquitin ligase activity of RING1B is not essential for early mouse development

et al. 2015

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Polycomb-repressive complex 1 (PRC1) and PRC2 maintain repression at many developmental genes in mouse embryonic stem cells and are required for early development. However, it is still unclear how they are targeted and how they function. We show that the ability of RING1B, a core component of PRC1, to ubiquitinate histone H2A is dispensable for early mouse embryonic development and much of the gene repression activity of PRC1. Our data support a model in which PRC1 and PRC2 reinforce each other's binding but suggest that the key functions of PRC1 lie beyond the enzymatic capabilities of RING1B.

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“…Nucleosomal DNAs were run on 1% agarose gels, and the mononucleosomal DNAs (150 bp) were gel-purified. Mononucleosomal DNAs from three digestions were pooled and prepared for sequencing (16).…”

Section: Methodsmentioning

confidence: 99%

BAF250a Protein Regulates Nucleosome Occupancy and Histone Modifications in Priming Embryonic Stem Cell Differentiation

Lei

West

Yan

et al. 2015

Journal of Biological Chemistry

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Background: How BAF250a regulates nucleosome configuration in ES cells is not clear. Results: BAF250a regulates nucleosome occupancy and H3K27me3 to control gene expression during ES cell differentiation. Conclusion: BAF250a plays a key role in poised chromatin regulation. Significance: Understanding the mechanisms of chromatin remodeling in poised chromatin regulation provides epigenetic insights into ES cell differentiation.

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Multiplexed Illumina sequencing libraries from picogram quantities of DNA

Cited by 79 publications

References 17 publications

Pseudomonas aeruginosa High-Level Resistance to Polymyxins and Other Antimicrobial Peptides Requires cprA , a Gene That Is Disrupted in the PAO1 Strain

Pseudomonas aeruginosa High-Level Resistance to Polymyxins and Other Antimicrobial Peptides Requires cprA , a Gene That Is Disrupted in the PAO1 Strain

The E3 ubiquitin ligase activity of RING1B is not essential for early mouse development

BAF250a Protein Regulates Nucleosome Occupancy and Histone Modifications in Priming Embryonic Stem Cell Differentiation

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