Notes: The authors extracted chloroplast DNA using a CTAB method and then cleaned up that DNA using the Wizard® DNA Clean-Up System before NGS on an Illumina Genome Analyzer II. (4916)

Notes: PCR products were analyzed on a 1% agarose gel and sized with molecular markers before selection for purification using the Wizard® SV Gel and PCR Clean-Up System. The chosen purified PCR amplicons were then used in NGS. (4557)

Notes: HepG2 cells were plated, 5 × 106 cells in 15cm plates. Transfections were performed 24 h later using Fugene HD. The authors used 15 μg of DNA per transfection and a Fugene:DNA ratio of 7:2. (4425)

Notes: The authors transiently transfected 5 × 10⁶ HEK293 cells per 150cm dish using the FuGENE® HD Transfection Reagent, 15µg of DNA and a reagent:DNA ratio of 7:2. (4432)

Notes: The authors extracted DNA from FFPE samples using the Maxwell® 16 Instrument and an unidentified Maxwell® FFPE DNA Kit. Purified DNA was subjected to NGS on an Illumina HiSeq 2000 Instrument. (4906)

Notes: These authors set out to determine how TET2 and TET3 proteins are involved in epigenetic regulation. To characterize proteins that interact with TET, the authors expressed full-length TET1, TET2 and TET3 as HaloTag® fusion proteins and performed protein pull-downs. They identified novel interactions between all three TET proteins and O-GlcNAc transferase (OGT), which catalyzes the addition of N-acetylglucosamine (GlcNAc) to numerous transcription factors, regulatory proteins and histones to activate or inhibit the target protein or recruit additional proteins. In this paper, they focused on TET2 and TET3, which showed the strongest interaction with OGT. They mapped TET2, TET3 and OGT binding throughout  the genome by expressing these proteins as HaloTag® fusion proteins in HEK293T cells, crosslinking the proteins and DNA, then capturing the fusion proteins and associated DNA fragments and performing high-throughput sequencing to show that TET2/3 and OGT colocalize at active gene promoters and were tightly clustered near transcription start sites.

For expression of HaloTag® fusion proteins and controls, HEK-293 cells were plated at 12 ×10⁶ cells in a 150mm dish and grown to 70–80% confluency before transfection with 30µg of plasmid using the FuGENE® HD Transfection Reagent.

To assess whether TET2/3-OGT activity affects the interaction of SET1/COMPASS with chromatin, the authors used bioluminescence resonance energy transfer (BRET). They created a fusion protein consisting of the H3K4 methyltransferase SETD1A and NanoLuc® luciferase as the energy donor and a fluorescently labeled histone H3.3-HaloTag® fusion protein as the energy acceptor.  These BRET experiments confirmed that TET2/3-OGT activity is necessary for SET1/COMPASS complex function and showed that TET and OGT activities promote binding of SETD1A, a component of the SET1/COMPASS complex, to chromatin. This binding increases H3K4me3 levels. Thus, the authors’ data support a TET2/3-OGT-mediated mechanism for regulating the SET1/COMPASS complex and thus H3K4me3. (4262)

Notes: DNA was extracted from 9 to 15 individual Wolbachia females either 2 or 8 days posteclosion, using the ReliaPrep™ gDNA Tissue MiniPrep System. Wolbachia density was then determined by relative qPCR. (4732)

Notes: These authors provide an overview of the advantages and disadvantages of techniques for transfection of primary neurons, and provide an optimized protocol for FuGENE-6 transfection of primary neuronal cultures. (4720)

Notes: Mouse Endothelial Fibroblast were isolated from transgenic mice and human LEDGF/p75 expression levels were examined by dye-based RT-qPCR. The total RNA was isolated with the ReliaPrep™ RNA Cell Miniprep System. (4605)

Notes: Total RNA was isolated from treated and untreated MC3T3-E1 mouse cells with the ReliaPrep™ RNA Cell Miniprep System and analyzed with an Agilent bioanalyzer. The isolated total RNA was labeled with Cy®3 dye and hybridized to an Agilent mouse array. (4615)

Notes: This paper describes a bioluminescence-based method for the detection of DNA methyltransferase activity based on methylation-resistant cleavage and protein expression. The authors used Dam methylase as a model enzyme, MboI as the methylation-resistant endonuclease, and luciferase reporter DNA (LR-DNA) as the target. LR-DNA was amplified by PCR and then used as substrate in a Dam methylation reaction. If the target sites in the LR-DNA were fully methylated, they were resistant to subsequent MboI cleavage and expressed luciferase upon in vitro transcription/translation. Incomplete methylation or the absence of methylation resulted in DNA digestion and diminished/absent luciferase activity. TNT® T7 Quick for PCR DNA was used for in vitro translation of the LR-DNA, and the Luciferase Assay System and GloMax® 20/20 Luminometer were used to measure luciferase activity. (4191)

Notes: Genomic DNA was isolated from rice plants and bisulfite treated. Target regions were specifically amplified using bisulfite primers, and the amplified products cloned into the pGEM®-T vector prior to large scale bisulfite sequencing. Sequencing analysis was performed using the Kismeth tool. pGEM®-T was also used as the vector for fragments amplified from cDNA clones of transcripts of interest prior to generating sense and antisense RNA probes for RNA in situ hybridization experiments. Apoptosis of tissue from anthers was assessed using the DeadEnd Fluorometric TUNEL System. (4558)

Notes: In this report, the original investigators who found XMRV and pMLV (polytropic murine leukemia virus) in blood of subjects with Chronic Fatigue Syndrome (CFS) report that this association is not confirmed in a blinded analysis of samples from rigorously characterized subjects.

The CDC performed nucleic acid testing assays. Plasma was centrifuged and RNA isolated from the pellet. Quantitative real-time RT-PCR assays (qRT-PCR) for generic pMLV/XMRV pro (protease) and gag detection were performed on RNA extracts, using the AccessQuick™ RT-PCR System and an AgPath one-step RT-PCR kit.

ArrayScript RT and AmpliTaq Gold DNA polymerase were used for cDNA synthesis and amplification in the pro and gag qRT-PCR assays, respectively. A third PCR was done using the primers XPOLOF and XPOLOR, followed by a nested PCR with the primers XPOLIF and XPOLIR for the generic detection of MLV/XMRV 216-bp pol sequences. For this reaction, cDNA synthesis and amplification of RNA was done using Promega AMV Reverse Transcriptase and a RobustI RT-PCR kit. Each PCR experiment included 20 water-only reactions to control for contamination. (4300)

Notes: These authors characterized a novel sulindac derivative (SBA) that inhibits cell proliferation and induces apoptosis in human colon tumor cells. To investigate the effect of SBA  on intracellular cGMP levels, they transfected HEK293 cells with a GloSensor™ construct containing firefly luciferase fused to the human PDE5 GAF-A cGMP binding domain (GloSensor cGMP-40F plasmid). Binding of cGMP causes a conformational change that results in increased luminescence. Kinetic measurements of luminescence showed that intracellular cGMP levels increased upon treatment with SBA. (4524)

Notes: These authors used the Roche 454 next generation sequencing platform to sequence and compare cancerous and normal horn tissue transcripts. mRNA isolated from each sample was fragmented prior to cDNA synthesis. cDNA quality was verified using a high sensitivity DNA Chip kit on the  Bioanalyzer 2100 and the  QuantiFluor™-ST Fluorometer. The transcripts were compared and potential tumor associated genes identified. (4229)

Notes: This paper describes a potential "specificity checkpoint" for nucleotide incorporation by DNA polymerases. Using Tgo DNA polymerase from Thermococcus gorgonarius as a model system, the authors identified a single key residue (E664) that influenced the ability to incorporate rNTPs. Mutation of E664 to lysine (K) enabled RNA polymerization in the context of another mutation (the steric-gate mutation (Y409G)). The paper describes characterization of the mutant phenotype. As part of the study, luciferase mRNA was generated from the luciferase control DNA using the mutant polymerase. Synthesis and in vitro translation of the synthesized RNA was also evaluated using ssDNA templates, New England Biolabs PURExpress™ protein synthesis kit, and the FluoroTect™ Green_Lys in vitro translation labeling system. (4248)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Algiphilus aromaticivorans. (4297)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Algoriphagus jejuensis. (4298)

Notes: These authors developed a protein-aggregation reporter that uses huntingtin exon 1 containing 72 glutamines fused to the N-terminal end of firefly luciferase (httQ72-Luc). This construct did not aggregate unless seeded by a non-luciferase-containing polyglutamine (polyQ) protein. Upon co-aggregation, httQ72-luc becomes insoluble and loses its enzymatic activity.  httQ72-Luc and a polyQ protein were used to screen a library for compounds that prevent polyQ aggregation. Leflunomide and its active metabolite teriflunomide inhibited protein aggregation independently of their known role in pyrimidine biosynthesis. This study demonstrated the usefulness of luciferase-based protein aggregate reporters for high-throughput screening applications. Luciferase activity was measured using the Luciferase Assay System and the GloMax®-Multi Detection System. (4188)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Anaerosalibacter bizertensis. (4308)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Enterococcus faecium. (4338)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Babesia spp. (4310)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Arthrobacter cupressi. (4309)

Notes: RAW 264.7 mouse macrophage cells were plated in 12-well plates at 1.5 × 10⁵ cells/well for transient transfection with 0.5μg of DNA and 1.5μl of FuGENE® 6 Transfection Reagent in 50µl of Opti-MEM I medium. After 24 hours, the cells were treated with IgG IC for 5 hours and luciferase activity analyzed using the Dual-Luciferase® Reporter Assay System. (4402)

Notes: Polyglutamine diseases are neurodegenerative disorders associated with the presence of proteins containing polyglutamine repeats. These authors studied the mechanism of polygluatmine toxicity. Mutant RNAs carrying an expanded CAG repeat were shown to activate the nucleolar stress pathway and induce apoptosis. Expanded CAG RNAs were shown to interact with nucleolin, preventing it from binding to an upstream control element of the rRNA promoter and causing decreased rRNA transcription, which in turn induced apoptosis. Perturbations in rRNA transcription were identified by real-time PCR, and fluorescence in situ hybridization was used to determine that expanded CAG RNAs localized to the nucleolus. Hybridization solutions were supplemented with RNasin® Ribonuclease Inhibitor. ImProm-II™ Reverse Transcriptase was used in RT-qPCR assays. (4228)