Journal: Frontiers in Microbiology

Article Title: Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses

doi: 10.3389/fmicb.2016.01588

Figure Lengend Snippet: Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including DNase digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.

Article Snippet: The following DNases were tested for their ability to remove amplifiable DNA from TNA samples: DNase I (Sigma), RNase-Free DNase Set (QIAGEN), RNase-Free DNase I (Epicentre Biotechnologies) and TURBO DNA-free DNase Kit (Ambion, Life Technologies).

Techniques: Environmental Sampling, Purification, Real-time Polymerase Chain Reaction, Lysis

Journal: Science Advances

Article Title: Matrix-bound nanovesicles within ECM bioscaffolds

doi: 10.1126/sciadv.1600502

Figure Lengend Snippet: Enzymatic digestion of decellularized ECM scaffolds releases small RNA molecules. ( A ) Nucleic acid extracted from untreated UBM (no digest) and pepsin-, proteinase K–, or collagenase-treated UBM was exposed to RNase A, DNase I, or no-nuclease treatment (control). ( B ) Electropherogram depicting the small RNA pattern of nucleic acid in fluorescence units (FU) before (top panel) and after (bottom panel) DNase I treatment. ( C ) Electropherogram depicting small RNA pattern from the indicated samples in FU. ( D ) A subset of nucleic molecules in biologic scaffolds is protected from nuclease degradation.

Article Snippet: RNase-free DNase was obtained from Qiagen.

Techniques: Fluorescence

Journal: Immunity

Article Title: Transformation of accessible chromatin and 3D nucleome underlies lineage commitment of early T cells

doi: 10.1016/j.immuni.2018.01.013

Figure Lengend Snippet: BCL11B binding is associated with an increase in chromatin interaction (A) Expression of Bcl11b from HSPC to DP from RNA-Seq analysis. (B) UCSC genome browser image showing the distribution of ChIP-Seq read density across the genomic region enclosing the Id2 locus (in red) for BCL11B binding, an active histone modification H3K27ac (two independent experiments), and a repressive histone modification H3K27me3, all in DP cells. Top track: distribution of DNase-Seq read density; Yellow and pink rectangles: BCL11B binding sites enriched with H3K27ac and H3K27me3, respectively; K.Z.: a representative BCL11B Chip-Seq data from Dr. Zhao’s lab, NHLBI (two independent experiments); E.V.R.: a representative BCL11B ChIP-Seq data from Prof. Rothenberg’s lab, Cal Tech (two independent experiments). (C) Gene Ontology enrichment analysis for genes with promoters bound by BCL11B and marked by repressive histone modification H3K27me3 in DP cells. (D) Observed versus expected number of genes, sorted based on the status of BCL11B binding and H3K27me3 marker at promoters and expression change by Bcl11b deletion in DP cells. Blue and red arrow heads: gene set repressed and activated by BCL11B, respectively. (E) Empirical cumulative distribution of the fold change of the number of TAD PETs from DN2 to DP cells for TADs sorted into four equal size groups based on the BCL11B coverage, defined by the percentage of genomic region bound by BCL11B in DP cells. P -value by K.-S. test. (F) WashU genome browser showing the distribution of BCL11B ChIP-Seq reads in DPs and the distribution of intra-TAD PETs in DN2 and DP cells for a 360K bps genomic region in chromosome 11. Red rectangle: TAD enriched with BCL11B binding and showing an increase in intra-TAD PETs; Green lines: TAD boundaries.

Article Snippet: Total RNA was extracted and on-column digestion with DNase (QIAGEN, Cat#79254) was performed, followed by elution with 10μl of RNase-free water.

Techniques: Binding Assay, Expressing, RNA Sequencing Assay, Chromatin Immunoprecipitation, Modification, Marker