Journal: PLoS ONE

Article Title: Assessing the Performance Capabilities of LRE-Based Assays for Absolute Quantitative Real-Time PCR

doi: 10.1371/journal.pone.0009731

Figure Lengend Snippet: Triplicate replicate amplifications of six quantities of lambda gDNA produce tight profile clustering, except for the two-molecule sample. Profile scattering implies loss of quantitative efficacy, a presumption supported by LRE quantification that generates target quantities that range from 1–4 molecules. Nevertheless, these LRE-based quantities produce an average of 2.3 molecules, which is close to the predicted target quantity. The numerical inlay summarizes the LRE analysis from which an average optical calibration factor (OCF) was derived.

Article Snippet: Lambda gDNA was obtained from New England BioLabs and diluted to the specified quantities with 10 mM Tris using siliconized microfuge tubes.

Techniques: Derivative Assay

Journal: PLoS ONE

Article Title: Assessing the Performance Capabilities of LRE-Based Assays for Absolute Quantitative Real-Time PCR

doi: 10.1371/journal.pone.0009731

Figure Lengend Snippet: Equal quantities of lambda gDNA were amplified in PCR reactions containing progressively greater volumes. ( A ) F C plots demonstrating that profile height is not only dependent on reaction volume, but that the concentration of amplicon DNA in the plateau phase (FU/µl) is similar for all reaction volumes. Predicted reaction fluorescence (circles) closely correlate with the actual fluorescence readings (dots). ( B ) LRE plots of the corresponding profiles demonstrates that increasing reaction volume has no impact on E max (Y intercept), but does produce a proportional increase in F max (X intercept). The numerical inlay provides a summary of the LRE analysis, which generated very similar F 0 values. The cycles included within the LRE window are designated by red circles. Vol, reaction volume; FU/µl, fluorescence units per µl of reaction at F max .

Article Snippet: Lambda gDNA was obtained from New England BioLabs and diluted to the specified quantities with 10 mM Tris using siliconized microfuge tubes.

Techniques: Amplification, Concentration Assay, Fluorescence, Generated

Journal: PLoS ONE

Article Title: Assessing the Performance Capabilities of LRE-Based Assays for Absolute Quantitative Real-Time PCR

doi: 10.1371/journal.pone.0009731

Figure Lengend Snippet: Equal quantities of lambda gDNA were amplified in which the time of annealing and elongation (A&E) was progressively reduced over four consecutive runs. ( A ) F C plots reveal a progressive reduction in F max along with changes in profile position and shape as E max was reduced. The predicted reaction fluorescence (circles) correlate well with the actual fluorescence readings (dots), further corroborating the ability of LRE to model PCR amplification. Nevertheless, it should be noted that loss of conformity within the plateau phase (referred to as plateau drift) is apparent at the two shortest A&E times, a trend that has been observed under other suboptimal assay conditions (data not shown). ( B ) LRE plots reveal little difference in ΔE (slope), with a progressive loss in F max (X intercept) as E max (Y intercept) is reduced, a trend that is very similar to the mathematical predictions shown in . The cycles included within the LRE window are designated by red circles.

Article Snippet: Lambda gDNA was obtained from New England BioLabs and diluted to the specified quantities with 10 mM Tris using siliconized microfuge tubes.

Techniques: Amplification, Fluorescence

Journal: PLoS ONE

Article Title: Assessing the Performance Capabilities of LRE-Based Assays for Absolute Quantitative Real-Time PCR

doi: 10.1371/journal.pone.0009731

Figure Lengend Snippet: Three datasets are presented, demonstrating that the fluorescence intensity generated by SYBR Green I is independent of reaction volume, GC content and amplicon size. Note that the mathematics of optical calibration is described in more detail in a previous study . ( A ) A known quantity of lambda gDNA is first amplified, from which an average F 0 value is determined (LRE-Fo). An optical calibration factor (OCF) is then calculated by dividing the average F 0 by the mass of the amplicon region derived from the lambda gDNA added to the reaction (M 0 ). OCF is thus expressed as fluorescence units per nanogram of dsDNA (FU/ng). For lambda gDNA, M 0 is calculated by multiplying the nanograms of lambda gDNA that are amplified by the amplicon size (A S ), and dividing by the genome size of lambda (48,502 bp). Overall, the similarity of the respective OCF values indicates that any difference in fluorescence intensity generated by these eight lambda amplicons is small. ( B ) To determine the number of target molecules within a sample (N 0 ), the process is reversed. M 0 is first calculated by dividing F 0 by an average OCF. N 0 is then calculated by multiplying M 0 by the number of base pairs per nanogram of dsDNA (9.1×10 11 bp/ng) and dividing by the amplicon size (A s ). For single-stranded DNA targets, such as cDNA, N 0 must be doubled as the OCF is derived from a double-stranded standard. The level of accuracy that can be achieved using an average OCF is illustrated by taking the F 0 values from A and comparing their respective N 0 values to the predicted number of target molecules, expressed here as percent error. Note that the reaction volume dataset that were taken from was produced using the same reaction setup and instrument used for the GC content and amplicon size datasets.

Article Snippet: Lambda gDNA was obtained from New England BioLabs and diluted to the specified quantities with 10 mM Tris using siliconized microfuge tubes.

Techniques: Fluorescence, Generated, SYBR Green Assay, Amplification, Derivative Assay, Produced

Journal: PLoS ONE

Article Title: Assessing the Performance Capabilities of LRE-Based Assays for Absolute Quantitative Real-Time PCR

doi: 10.1371/journal.pone.0009731

Figure Lengend Snippet: Amplicon primer sequences.

Article Snippet: Lambda gDNA was obtained from New England BioLabs and diluted to the specified quantities with 10 mM Tris using siliconized microfuge tubes.

Techniques: Amplification

Journal: bioRxiv

Article Title: Opposing transcription factors MYCL and HEY1 mediate the Notch-dependent airway stem cell fate decision

doi: 10.1101/2022.10.05.511009

Figure Lengend Snippet: A) mTECs transduced with MYCL-GFP, HEY1-GFP or a control (NLS-GFP) lentivirus were analyzed using CUT&RUN after one day at air/liquid interface. Distribution of MYCL and HEY1 peaks relative to gene positions. B) Venn diagram of the overlap of MYCL and HEY1 binding sites in mTECs. C) MYCL, HEY1 and control CUT&RUN signals near the Gmnc , Mcidas, Foxj1 , and Ccno loci. Gene models are depicted 5’ to 3’. Regions with identified peaks are outlined in black. Scale bars depict 1 kilobase pairs of DNA. D) qRT-PCR measurement of Gmnc , Mcidas, Foxj1 and Ccno expression in control (NLS-GFP) MYCL-GFP transduced mTECs cultured for one day at air/liquid interface. Significance was assessed using a two-way ANOVA test with multiple comparison correction, with *** indicating p = 0.0009 and **** indicating p < 0.0001. Error bars represent SEM for 3 replicates of independently derived and transduced mTECs. E) Multiplexed RNA-FISH and immunofluorescence of control (NLS-GFP) or MYCL-GFP transduced mTECs cultured for one day at air/liquid. Gmnc and Mcidas staining was multiplexed with immunostaining for CDH1 to mark cell boundaries.

Article Snippet: Library preparation was performed with the NEB Next Ultra II DNA Library Prep Kit for Illumina (NEB, Cat #E7645S) using 25 μL of CUT&RUN DNA as input, as detailed by Liu N. ( https://www.protocols.io/view/library-prep-for-cut-amp-run-with-nebnext-ultra-ii-kxygxm7pkl8j/v2 ).

Techniques: Transduction, Binding Assay, Quantitative RT-PCR, Expressing, Cell Culture, Derivative Assay, Immunofluorescence, Staining, Immunostaining

Journal: bioRxiv

Article Title: Synthetic genomic reconstitution reveals principles of mammalian Hox cluster regulation

doi: 10.1101/2021.07.07.451065

Figure Lengend Snippet: (A) Layout of rat HoxA locus in the rn6 genome assembly. The rn6 genome includes an erroneous duplication at the HoxA locus between gaps in the assembly. The SynHoxA assemblon sequence is based on bringing together the two ‘separate’ RnHoxA segments. The sequence was segmented into 28 ∼5kb PCR amplicons with terminal homology of ∼200bp to adjacent amplicons. Conservation to the mouse genome is depicted using the multiz track from the UCSC genome browser. (B) Schematic depicting the assembly workflow for the 134kb SynHoxA assemblon. BACs containing Rat HoxA were used as PCR template to generate 28 segments tiling the entire HoxA locus. These segments were co-transformed into yeast with appropriate linkers and assembly vector to build two ∼65kb half assemblons into centromeric yeast-bacteria shuttle vectors. These half assemblons are recovered to bacteria and amplified. Full 134kb assemblon was built from half assemblons after releasing them from the vector using terminal restriction enzymes ( AsiSI ) and transforming into yeast. Full assemblon was then recovered from yeast into bacteria for amplification and verification. (C) Agarose gel of the 28 PCR amplicons that tile the 134kb SynHoxA assemblon. (D) Strategy to PCR-screen yeast colonies derived from assembly experiments. Primers (red arrows) span assembly junctions and test presence/absence of amplicons in many yeast colonies. Reproduced from ref with permission from authors. (E) Agarose gel showing one yeast colony carrying the full 134kb SynHoxA assemblon verified manually for the presence of all assembly junctions, using the strategy outlined in panel D. (F) Half and Full 134kb SynHoxA assemblon BACs purified from E.coli were digested with PvuI and separated using field inversion gel electrophoresis (FIGE). Lambda monocut ladder sizes are indicated in kb. Band sizes correspond to expected fragments.

Article Snippet: 500 ng of lambda monocut ladder (New England Biolabs N3019S) were used as a molecular weight standard.

Techniques: Sequencing, Transformation Assay, Plasmid Preparation, Amplification, Agarose Gel Electrophoresis, Derivative Assay, Purification, Nucleic Acid Electrophoresis

Journal: bioRxiv

Article Title: Synthetic genomic reconstitution reveals principles of mammalian Hox cluster regulation

doi: 10.1101/2021.07.07.451065

Figure Lengend Snippet: (A) Layout of rat HoxA locus from the rn6 genome assembly depicting genes, Rn HoxA cluster segments in black and previously identified distal enhancers in purple. The Enhancers+SynHoxA assemblon sequence is made by stringing all the enhancers directly upstream of the SynHoxA assemblon sequence. Conservation to mouse genome is depicted using multiz track from the UCSC genome browser. (B) PCR amplicons tiling enhancer sequences were generated from Rat HoxA BACs and co-transformed into a yeast strain containing the 134kb SynHoxA assemblon with a gRNA vector targeting the left terminus of the 134kb assemblon. The enhancer PCR amplicons were used to repair this break, resulting in the construction of the 170kb Enhancers+SynHoxA assemblon. Assemblon was recovered into bacteria for amplification and verification. (C) Agarose gel of the 8 PCR amplicons containing enhancer sequences. (D) Agarose gel showing one yeast colony tested for the presence of novel enhancer assembly junctions and with primers spanning 134kb SynHoxA . (E) 134kb and 170kb assemblon BACs purified from E.coli were digested with PvuI and separated using FIGE. Lambda monocut ladder sizes are indicated in kb. Band sizes correspond to expected fragments.

Article Snippet: 500 ng of lambda monocut ladder (New England Biolabs N3019S) were used as a molecular weight standard.

Techniques: Sequencing, Generated, Transformation Assay, Plasmid Preparation, Amplification, Agarose Gel Electrophoresis, Purification

Journal: bioRxiv

Article Title: Synthetic genomic reconstitution reveals principles of mammalian Hox cluster regulation

doi: 10.1101/2021.07.07.451065

Figure Lengend Snippet: (A) Schematic of assembly strategy for 130kb RAREΔ SynHoxA and 166kb Enhancers + RAREΔ SynHoxA . Nature of the RARE mutations is shown on the right. RAR binding data comes from previously published reports. (see Methods) (B) Sanger sequencing traces confirmed precise CRISPR editing of RAREs in yeast. (C) SynHoxA assemblon BACs purified from E.coli were digested with PvuI and separated using FIGE. Lambda monocut ladder sizes are indicated in kb. Bands correspond to expected fragment lengths. (D) Sequencing data of assemblon BACs purified from E. coli aligned to a custom mm10 reference genome. Positions of the enhancers and protein coding genes are shown in black.

Article Snippet: 500 ng of lambda monocut ladder (New England Biolabs N3019S) were used as a molecular weight standard.

Techniques: Binding Assay, Sequencing, CRISPR, Purification