Journal: The Plant journal : for cell and molecular biology

Article Title: The PPR-SMR protein PPR53 enhances the stability and translation of specific chloroplast RNAs in maize.

doi: 10.1111/tpj.13093

Figure Lengend Snippet: Figure 5. Analysis of chloroplast rRNAs in ppr53 mutants. (a) RNA gel blot analysis of transcripts from the rrn transcription unit. Replicate blots of seedling leaf RNA were hybridized to the probes indicated on the map. Three heteroallelic ppr53 individuals with distinct pigment phenotypes (iv vir, vpyg vir and vir; see Figure 1) were analyzed. RNAs from the non-photosynthetic mutants atp4 and wtf2 were analyzed for comparison. The wtf2 mutant is shown to illustrate pleiotropic effects resulting from the loss of plastid ribosomes. An image of one of the blots stained with methylene blue is shown below to illustrate equal loading of cytosolic 28S and 18S rRNAs. (b) Primer extension assay to quantify processed transcripts with a 50-end mapping 70 nucleotides upstream of rrn23. A 50-end labeled 24-nucleotide primer starting four nucleotides upstream of mature 23S rRNA was used to prime reverse transcription on 5 lg of seedling leaf RNA. The ribosome-deficient mutant wtf2 (see panel a) as well as two other non-photosynthetic mutants (atp4 and crp1) accumulate increased levels of the 70 23S rRNA precursor (see arrow), whereas it is undetectable in all three ppr53 mutant individuals. The abundance of longer rrn23 processing intermediates is similar in all mutant samples ana- lyzed, as shown also on the RNA gel blot in panel (a) (see probe 2 data). (c) PPR53-dependent sRNA mapping to the 50-end of the PPR53-dependent rrn23 precursor. Screen captures from the Integrated Genome Viewer show reads as pink lines and a histogram of read counts in gray (above). The 50-end of the PPR53-dependent sRNAs corresponds to that of the PPR53-dependent pre-23S rRNA. The sRNA reads from flanking regions serve as internal standards. Data from a ppr4 mutant are shown to control for effects resulting from the loss of plastid ribosomes. The loss of this PPR53-dependent sRNA is accompanied by an increase in the abundance of an sRNA with a 50-end two nucleotides upstream. The basis for this effect is unknown.

Article Snippet: Sequencing libraries were prepared with the NEBNext Multiplex Small RNA Library Prep Set.

Techniques: Western Blot, Comparison, Mutagenesis, Staining, Primer Extension Assay, Labeling, Reverse Transcription, Control

Journal: Cell

Article Title: Transmission from vaccinated individuals in a large SARS-CoV-2 Delta variant outbreak

doi: 10.1016/j.cell.2021.12.027

Figure Lengend Snippet: Epidemiology of the COVID-19 Provincetown outbreak and overview of the dataset (A) SARS-CoV-2 genomes in this dataset by collection date. (B) Distribution of all cases in Barnstable County (gray bars) and estimate of reproductive number ( R t ) (red line) over the course of the outbreak. (C) Flow diagram of sample sets presented here. Gray shading indicates sample sets reported and analyzed in this study. (D) Cycle threshold (Ct) value of the N1 gene for the 313 individuals known to be fully vaccinated by BNT162b2, mRNA-1273, or AD26.CoV2.S; includes linear regression with 95% confidence interval. All Ct values were collected using the same platform and assay. See also Figure S1 .

Article Snippet: Illumina sequencing libraries were prepared using the NEBNext ARTIC v3 SARS-CoV-2 FS Library Prep Kit.

Techniques:

Journal: Cell

Article Title: Transmission from vaccinated individuals in a large SARS-CoV-2 Delta variant outbreak

doi: 10.1016/j.cell.2021.12.027

Figure Lengend Snippet: Genomic epidemiology and limited onward impact of the Provincetown outbreak (A) Time tree of SARS-CoV-2 outbreak-associated genomes (blue dots) in the global context. Inset, frequencies of Pango lineages among outbreak-associated genomes. An interactive version of this tree is available at: https://auspice.broadinstitute.org/sars-cov-2/ma-delta/20211005/cluster-unique-usher . (B) Percentage of all Delta-lineage genomes from MA detected by baseline genomic surveillance with the mutational signature of the dominant outbreak cluster (red line). Percentages are calculated and shown per day based on sample collection date. Dashed line: cumulative new cases in MA over the same period (available at: https://www.mass.gov/info-details/covid-19-response-reporting ). (C) Numbers of genomes (left) and percentage of Delta-lineage genomes (right) per state that are estimated to descend from the largest cluster in the Provincetown outbreak. Barplots show lower-bound (solid) and upper-bound (faded) estimates, calculated as described in STAR Methods. Note that the scale of the x axis in the plot on the right is in percentages, with a maximum of 1%. See also and ; .

Article Snippet: Illumina sequencing libraries were prepared using the NEBNext ARTIC v3 SARS-CoV-2 FS Library Prep Kit.

Techniques:

Journal: Cell

Article Title: Transmission from vaccinated individuals in a large SARS-CoV-2 Delta variant outbreak

doi: 10.1016/j.cell.2021.12.027

Figure Lengend Snippet: Outbreak introductions and onward spread, related to (A) Left: time tree of outbreak-associated SARS-CoV-2 genomes in a global context colored by association with the Provincetown outbreak (as in A). Right: each introduction into Provincetown as inferred from the phylogenetic tree based on a change in ancestral inference of a branch to “outbreak-associated.” Gray dots represent the most recent common ancestor of the clade that was inferred to be from outside of Provincetown. All outbreak-associated samples downstream of each node are shown in the full phylogenetic tree. (B) The percentage of all Delta-lineage baseline surveillance genomes from MA with the mutational signature of the five Delta-lineage clusters among outbreak-associated cases. Three mutations (C8752T, C20451T, and A26759G) are shared by the majority of all outbreak-associated genomes, referred to as the dominant cluster. The remaining four clusters were defined by their characteristic mutations, with cluster 2 defined by G4124A and A5608G; cluster 3 by T7858C and A29257C; cluster 4 by T23131C; and cluster 5 by A26759G, C20451T, C7600T, and T27940C. Percentages per day based on sample collection date and new cumulative cases in MA were plotted over time from July 3rd to August 31st, 2021.

Article Snippet: Illumina sequencing libraries were prepared using the NEBNext ARTIC v3 SARS-CoV-2 FS Library Prep Kit.

Techniques:

Journal: Cell

Article Title: Transmission from vaccinated individuals in a large SARS-CoV-2 Delta variant outbreak

doi: 10.1016/j.cell.2021.12.027

Figure Lengend Snippet: Genomic support for transmission links and transmission predictions by vaccination status and symptoms, related to Figure 3 (A) Maximum-likelihood phylogenetic tree of the only high-confidence transmission pair from contact tracing without strong statistical support in outbreaker2 transmission reconstruction. This pair was in a cluster of six identical consensus genomes with very similar collection dates. No symptom onset date was known for either of the individuals in the pair. Even when incorporating contact tracing information into the model, another sample was predicted as almost equally likely to have been the ancestor of this case. (B) Maximum-likelihood phylogeny of the large cluster of cases associated with a single index case in a close-contact setting. Cases from this epidemiological cluster are colored in dark red. (A) and (B) are part of a larger phylogenetic tree available at https://auspice.broadinstitute.org/sars-cov-2/ma-delta/20211005/cluster-unique-usher . (C) Gray bars, fractions, and 90% confidence intervals indicate the proportion of individuals that were the origin of at least one transmission event predicted by outbreaker2 with a probability of >70%. Individuals are separated by vaccination status (left), days from final vaccination date to collection date (middle), or presence or absence of symptoms in vaccinated individuals (right). 90% binomial confidence intervals were calculated using the exact method through the binom package in R. Using simulations incorporating outbreaker2’s confidence in putative transmission links, we calculate that an infected unvaccinated individual was 0.18–2.11 times as likely to transmit as an infected vaccinated individual. Among fully vaccinated individuals, an infected asymptomatic individual was 0–0.99 times as likely to transmit as an infected symptomatic individual. Our estimates of relative risk are predicated on outbreaker2 correctly estimating the probability that it has chosen the correct index case of each putative transmission.

Article Snippet: Illumina sequencing libraries were prepared using the NEBNext ARTIC v3 SARS-CoV-2 FS Library Prep Kit.

Techniques: Transmission Assay, Infection

Journal: Cell

Article Title: Transmission from vaccinated individuals in a large SARS-CoV-2 Delta variant outbreak

doi: 10.1016/j.cell.2021.12.027

Figure Lengend Snippet:

Article Snippet: Illumina sequencing libraries were prepared using the NEBNext ARTIC v3 SARS-CoV-2 FS Library Prep Kit.

Techniques: Isolation, Quantitative RT-PCR, Diagnostic Assay, RNA Sequencing Assay, Software

Journal: Journal of Advanced Research

Article Title: A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries

doi: 10.1016/j.jare.2019.12.005

Figure Lengend Snippet: Comparison of human rRNA removal specificity and efficiency between the RTR2D procedure and NEBNext® rRNA Depletion kit. Human total RNA (1.0 µg) was subjected to the RTR2D (R2D) (with the pooled rRNA probes) or the NEBNext® rRNA Depletion (NEB) protocol. The rRNA-depleted products were subjected to the Bioanalyzer. Representative gel image ( a ) and electropherograms ( b ) are shown. “Input” and “No probes (NP)” groups were used as controls. ( B ) TqPCR analysis of the residual rRNA species ( a ) and removal efficiency ( b ). “**” p < 0.001, compared with that of the NP group or the Input group. ( C ) Effect of rRNA removal protocols on the expression of housekeeping genes GAPDH and β-ACTIN ( a ) and genes/lncRNA with different abundances, c-MYC , TP53 and lncRNA HOTAIR ( b ). All qPCR reactions were done in triplicate.

Article Snippet: Commercially available pull-out kits include Ribo-Zero (Illumina, USA) and RiboMinus (Thermo Fisher, USA), while the RNase H-based rRNA degradation of oligo-DNA:RNA hybrids includes RiboErase (Kapa Biosystems, USA) and NEBNext rRNA Depletion (New England Biolabs, NEB), which depletes both cytoplasmic (5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA) and mitochondrial ribosomal RNA (12S rRNA and 16S rRNA) from human, mouse and rat total RNA preparations.

Techniques: Expressing

Journal: Journal of Advanced Research

Article Title: A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries

doi: 10.1016/j.jare.2019.12.005

Figure Lengend Snippet: Comparison of mouse rRNA removal specificity and efficiency between the RTR2D procedure and NEB Next rRNA Depletion kit. Mouse total RNA (1.0 µg) was subjected to the RTR2D (R2D) or the NEBNext® rRNA Depletion (NEB) protocol. The rRNA-depleted products were subjected to the Bioanalyzer. Representative gel image ( a ) and electropherograms ( b ) are shown. “Input” and “No probes (NP)” groups were used as controls. ( B ) TqPCR analysis of the residual rRNA species ( a ) and removal efficiency ( b ). “**” p < 0.001, compared with that of the NP group or the Input group. ( C ) Effect of rRNA removal protocols on the expression of housekeeping genes Gapdh and β-Actin ( a ) and genes/lncRNA with different abundances, c-Myc , Tp53 and lncRNA Hotair ( b ). All qPCR reactions were done in triplicate.

Article Snippet: Commercially available pull-out kits include Ribo-Zero (Illumina, USA) and RiboMinus (Thermo Fisher, USA), while the RNase H-based rRNA degradation of oligo-DNA:RNA hybrids includes RiboErase (Kapa Biosystems, USA) and NEBNext rRNA Depletion (New England Biolabs, NEB), which depletes both cytoplasmic (5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA) and mitochondrial ribosomal RNA (12S rRNA and 16S rRNA) from human, mouse and rat total RNA preparations.

Techniques: Expressing

Journal: Journal of Advanced Research

Article Title: A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries

doi: 10.1016/j.jare.2019.12.005

Figure Lengend Snippet: Transcriptomic comparison of the RNA-seq libraries prepared with the RTR2D and the NEBNext rRNA Depletion protocols. Human total RNA (1.0 µg) was subjected to the rRNA removal process by using the RTR2D procedure (R2D) and the NEBNext rRNA Depletion kit (NEB). The rRNA-depleted samples were used for RNA-seq library preparations using the Illumina protocol and subjected to NGS analysis (in replicate). ( A ) The average valid reads (RPKM) for the RTR2D ( a ) and the NEB kit ( b ) are depicted in various categories of transcripts. ( B ) Scatter plots and correlations of mRNA transcripts between the RTR2D and the NEB protocols in two different batches of preparations ( a & b ). ( C ) Scatter plots and correlations of lncRNA transcripts between the RTR2D and the NEB protocols in two different batches of preparations ( a & b ).

Article Snippet: Commercially available pull-out kits include Ribo-Zero (Illumina, USA) and RiboMinus (Thermo Fisher, USA), while the RNase H-based rRNA degradation of oligo-DNA:RNA hybrids includes RiboErase (Kapa Biosystems, USA) and NEBNext rRNA Depletion (New England Biolabs, NEB), which depletes both cytoplasmic (5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA) and mitochondrial ribosomal RNA (12S rRNA and 16S rRNA) from human, mouse and rat total RNA preparations.

Techniques: RNA Sequencing Assay

Journal: Journal of Advanced Research

Article Title: A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries

doi: 10.1016/j.jare.2019.12.005

Figure Lengend Snippet: Nutlin3A-induced transcriptomic changes determined by RNA-seq analysis of the NGS libraries prepared with the RTR2D and the NEBNext rRNA Depletion protocols . Exponentially growing human osteosarcoma line SJSA1 cells were treated with 2 µM Nutlin3A or DMSO control for 24 h and subjected to total RNA isolation. Human total RNA (1.0 µg) was subjected to rRNA removal by using the RTR2D procedure (R2D) and the NEBNext rRNA Depletion kit (NEB). The rRNA-depleted samples were used for RNA-seq library preparations using the Illumina protocol and subjected to NGS analysis. ( A ) Scatter plots of mRNA transcripts affected by DMSO (Y-axis) and Nutlin3A (X-axis) identified in the RNA-seq libraries prepared with the NEB protocol ( a ) or the RTR2D protocol ( b ). Venn diagrams were generated by using more stringent criteria for the differentially expressed transcripts for up-regulated ( c ) (total relative reads > 54) and down-regulated transcripts ( d ) (total relative reads > 13). ( B ) Scatter plots of lncRNA transcripts affected by DMSO (Y-axis) and Nutlin3A (X-axis) identified in the RNA-seq libraries prepared with the NEB protocol ( a ) or the RTR2D protocol ( b ). Significant up and down-regulated transcripts were defined as log2FoldChange > 1 and < −1, respectively, with a false detection rate (FDR) < 0.001. Venn diagrams were generated by using more stringent criteria for the differentially expressed transcripts for up-regulated ( c ) (total relative reads > 540) and down-regulated transcripts ( d ) (total relative reads > 20).

Article Snippet: Commercially available pull-out kits include Ribo-Zero (Illumina, USA) and RiboMinus (Thermo Fisher, USA), while the RNase H-based rRNA degradation of oligo-DNA:RNA hybrids includes RiboErase (Kapa Biosystems, USA) and NEBNext rRNA Depletion (New England Biolabs, NEB), which depletes both cytoplasmic (5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA) and mitochondrial ribosomal RNA (12S rRNA and 16S rRNA) from human, mouse and rat total RNA preparations.

Techniques: RNA Sequencing Assay, Isolation, Generated