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

Journal: bioRxiv

Article Title: FFPE DNA shows two major error profiles derived from deamination of cytosine and methylcytosine that can be mitigated using distinct repair strategies

doi: 10.1101/2023.03.02.530819

Figure Lengend Snippet: A . Overall percentage of CG:TA transitions in sequencing reads after library amplification using Q5 (left), Q5U® (center) and Taq DNA polymerase (right), with (DNA repair (orange), UDG (brown)) and without (blue) DNA repair treatment of the lung tumor FFPE sample. Experiments were done in duplicate and error bars are reflecting the variability between replicates. B . Profiles of C to T transitions on read 1 (R1, left) and read 2 (R2, right) as a function of read position (in bp). Libraries were amplified using Q5 (top), Q5U® (middle) and Taq DNA polymerase (bottom). Experiments were done in duplicate. Fraction of C to T is a global count of the of C (on the reference genome) to T (on the mapped reads) normalized to the total number of C to C matches. C . Schema representing the fate of the deaminated strand (gray) and the un-damaged strand (purple) after end repair (red) and amplification with Q5 (left) and Taq or Q5U (right).

Article Snippet: The DNA repair step commonly used in this manuscript corresponds to the NEBNext FFPE DNA Repair v2 Module and contains enzymes and buffers that are optimized to repair FFPE DNA in next generation sequencing workflow including UDG, Endonuclease IV, Fpg, Endonuclease VIII, T4 PDG, Taq DNA ligase and Bst DNA polymerase.

Techniques: Sequencing, Amplification

Journal: bioRxiv

Article Title: FFPE DNA shows two major error profiles derived from deamination of cytosine and methylcytosine that can be mitigated using distinct repair strategies

doi: 10.1101/2023.03.02.530819

Figure Lengend Snippet: A . Overall percentage of CG:TA transitions in sequencing reads for lung (left) and pancreatic (right) tumor FFPE samples. Samples were either left untreated (orange) or treated with DNA repair mix before (red) and after (blue) end repair. B . Breakdown of the fraction of C:T transitions according to Read types, Read 1 (R1, top) and Read 2 (R2, bottom) and position on the read starting from the 5’ end. Two FFPE samples have been used for these experiments : Lung tumor FFPE sample (Blue) and pancreatic tumor FFPE sample (red) treated with DNA repair after end repair (left), before end repair (right) and no treatment (middle). Fraction of C to T is a global count of the of C (on the reference genome) to T (on the mapped reads) normalized to the total number of C to C matches. Red arrows indicate positions of increased C to T transition rate.

Article Snippet: The DNA repair step commonly used in this manuscript corresponds to the NEBNext FFPE DNA Repair v2 Module and contains enzymes and buffers that are optimized to repair FFPE DNA in next generation sequencing workflow including UDG, Endonuclease IV, Fpg, Endonuclease VIII, T4 PDG, Taq DNA ligase and Bst DNA polymerase.

Techniques: Sequencing

Journal: bioRxiv

Article Title: FFPE DNA shows two major error profiles derived from deamination of cytosine and methylcytosine that can be mitigated using distinct repair strategies

doi: 10.1101/2023.03.02.530819

Figure Lengend Snippet: Substitution profiles for low (less than 1% A ) and high (more than 10% B ) allelic frequency substitution. Barplot representing the number of genomic positions per megabases (mb). The barplots are color coded according to the substitution types and organized by contexts (XpA, XpT, XpC and XpG with X= A, C, T or G) and reads (read1, top and read2, bottom). Sequencing depth has been downsample to 300-fold coverage for comparison, thus the variant read number for high (>10%) allelic frequency is above 30 reads supporting the presence of a variant and low (<1%) allelic frequency is below 3 reads supporting the presence of a variant. The equivalent result for medium (1 to 10%) allelic frequency substitution is plotted on and . All reads substitution have a Phred Quality Scores Q>30. CpA context has no high allelic frequency substitution in Read 2 in the DNA repair sample. The excess of G to T can be attributed to oxidative damage.

Article Snippet: The DNA repair step commonly used in this manuscript corresponds to the NEBNext FFPE DNA Repair v2 Module and contains enzymes and buffers that are optimized to repair FFPE DNA in next generation sequencing workflow including UDG, Endonuclease IV, Fpg, Endonuclease VIII, T4 PDG, Taq DNA ligase and Bst DNA polymerase.

Techniques: Sequencing, Variant Assay