Journal: Nucleic Acids Research

Article Title: Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs

doi: 10.1093/nar/gku118

Figure Lengend Snippet: Viral RNA fragments produced by RNase L and RNase A. HCV and PV RNAs were incubated with RNase L and RNase A to produce RNA fragments for 2′, 3′-cyclic phosphate cDNA synthesis and sequencing. Agarose gel electrophoresis and ethidium bromide staining revealed the size of viral RNA fragments. ( A ) Diagram of HCV and PV RNAs. HCV RNA is 9648 bases long. PV RNA is 7500 bases long. ( B ) Viral RNAs incubated with RNase L. HCV and PV RNAs were incubated with RNase L for 20 min in the absence of 2-5A (no 2-5A), or with RNase L and 2-5A for 0, 2.5, 5, 10 and 20 min. ( C ) Viral RNAs incubated with RNase A. HCV and PV RNAs were incubated for 20 min in the absence of RNase A (−), and the presence of RNase A for 0, 2.5, 5, 10 and 20 min.

Article Snippet: When RNAs from the PV-infected cells were analyzed by agarose gel electrophoresis, the accumulation of viral RNA was evident at 4–8 h post adsorption (hpa) ( B). rRNA fragments characteristic of RNase L activity were evident in RNAs from PV-infected W12 HeLa cells at 6 and 8 hpa, but these rRNA fragments were not detected in PV-infected M25 HeLa cells ( B, asterisks indicate the location of rRNA fragments characteristic of RNase L activity). cDNA libraries were prepared and sequenced using the RNAs from HeLa cells ( C and D and Supplementary Table S3 ). cDNA reads corresponding to PV RNA increased from undetectable levels at early times after infection to 5% of the cDNA at 6 hpa in W12 HeLa cells and 26.1% of the cDNA at 6 hpa in M25 HeLa cells ( C and D and Supplementary Table S3 ).

Techniques: Produced, Incubation, Sequencing, Agarose Gel Electrophoresis, Staining

Journal: Nucleic Acids Research

Article Title: Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs

doi: 10.1093/nar/gku118

Figure Lengend Snippet: Endoribonuclease cleavage sites in PV RNA isolated from HeLa cells. RNAs from PV-infected HeLa cells ( Figure 3 B) were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. ( A ) Location and frequency of cleavage sites in PV RNA from W12 HeLa cells. X-axis: Nucleotide position in PV RNA. Y-axis: Number of distinct UMI-tagged linkers detected at each cleavage site. ( B ) Dinucleotide specificity of cleavage sites in PV RNA from W12 HeLa cells. X-axis: Dinucleotide at the 3′-end of PV RNA fragments (adjacent to 8 base UMI sequence in RNA linkers as illustrated in Supplementary Figure S1 ). Y-axis: Percent of PV cDNA reads. ( C ) Location and frequency of cleavage sites in PV RNA from M25 HeLa cells. X-axis: Nucleotide position in PV RNA. Y-axis: Number of distinct UMI-tagged linkers detected at each cleavage site. ( D ) Dinucleotide specificity of cleavage sites in PV RNA from M25 HeLa cells. X-axis: Dinucleotide at the 3′-end of PV RNA fragments. Y-axis: Percent of PV cDNA reads.

Article Snippet: When RNAs from the PV-infected cells were analyzed by agarose gel electrophoresis, the accumulation of viral RNA was evident at 4–8 h post adsorption (hpa) ( B). rRNA fragments characteristic of RNase L activity were evident in RNAs from PV-infected W12 HeLa cells at 6 and 8 hpa, but these rRNA fragments were not detected in PV-infected M25 HeLa cells ( B, asterisks indicate the location of rRNA fragments characteristic of RNase L activity). cDNA libraries were prepared and sequenced using the RNAs from HeLa cells ( C and D and Supplementary Table S3 ). cDNA reads corresponding to PV RNA increased from undetectable levels at early times after infection to 5% of the cDNA at 6 hpa in W12 HeLa cells and 26.1% of the cDNA at 6 hpa in M25 HeLa cells ( C and D and Supplementary Table S3 ).

Techniques: Isolation, Infection, Sequencing

Journal: Nucleic Acids Research

Article Title: Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs

doi: 10.1093/nar/gku118

Figure Lengend Snippet: Endoribonuclease cleavage sites in rRNAs from M25 HeLa cells. RNAs from mock-infected and PV-infected M25 HeLa cells ( Figure 3 B) were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. The location and frequency of cleavage sites in 28S rRNA ( A ), 18S rRNA ( B ), 5.8S rRNA ( C ) and 5S rRNA ( D ) are shown for mock-infected and PV-infected RNA samples isolated at 8 hpa. X-axis: Nucleotide position of each RNA. Y-axis: Percentage of total UMIs at each cleavage site. Dinucleotides at the 3′-end of abundant RNA fragments are annotated at the corresponding positions in the graphs. The locations of GC-rich expansion segments are highlighted by light blue rectangles.

Article Snippet: When RNAs from the PV-infected cells were analyzed by agarose gel electrophoresis, the accumulation of viral RNA was evident at 4–8 h post adsorption (hpa) ( B). rRNA fragments characteristic of RNase L activity were evident in RNAs from PV-infected W12 HeLa cells at 6 and 8 hpa, but these rRNA fragments were not detected in PV-infected M25 HeLa cells ( B, asterisks indicate the location of rRNA fragments characteristic of RNase L activity). cDNA libraries were prepared and sequenced using the RNAs from HeLa cells ( C and D and Supplementary Table S3 ). cDNA reads corresponding to PV RNA increased from undetectable levels at early times after infection to 5% of the cDNA at 6 hpa in W12 HeLa cells and 26.1% of the cDNA at 6 hpa in M25 HeLa cells ( C and D and Supplementary Table S3 ).

Techniques: Infection, Sequencing, Isolation

Journal: Nucleic Acids Research

Article Title: Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs

doi: 10.1093/nar/gku118

Figure Lengend Snippet: Frequency, location and dinucleotide specificity of cleavage sites in viral RNAs. 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing was used to analyze the viral RNA fragments shown in Figure 1 . ( A ) Frequency, location and dinucleotide specificity of endoribonuclease cleavage sites in HCV RNA (from 20 min samples). ( B ) Frequency, location and dinucleotide specificity of cleavage sites in PV RNA (from 20 min samples).

Article Snippet: When RNAs from the PV-infected cells were analyzed by agarose gel electrophoresis, the accumulation of viral RNA was evident at 4–8 h post adsorption (hpa) ( B). rRNA fragments characteristic of RNase L activity were evident in RNAs from PV-infected W12 HeLa cells at 6 and 8 hpa, but these rRNA fragments were not detected in PV-infected M25 HeLa cells ( B, asterisks indicate the location of rRNA fragments characteristic of RNase L activity). cDNA libraries were prepared and sequenced using the RNAs from HeLa cells ( C and D and Supplementary Table S3 ). cDNA reads corresponding to PV RNA increased from undetectable levels at early times after infection to 5% of the cDNA at 6 hpa in W12 HeLa cells and 26.1% of the cDNA at 6 hpa in M25 HeLa cells ( C and D and Supplementary Table S3 ).

Techniques: Sequencing

Journal: Nucleic Acids Research

Article Title: Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs

doi: 10.1093/nar/gku118

Figure Lengend Snippet: Endoribonuclease cleavage sites in rRNAs from W12 HeLa cells. RNAs from mock-infected and PV-infected W12 HeLa cells ( Figure 3 B) were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. The location and frequency of cleavage sites in 28S rRNA ( A ), 18S rRNA ( B ), 5.8S rRNA ( C ) and 5S rRNA ( D ) are shown for mock-infected and PV-infected RNA samples isolated at 8 hpa. X-axis: Nucleotide position of each RNA. Y-axis: Percentage of total UMIs at each cleavage site. Dinucleotides at the 3′-end of abundant RNA fragments are annotated at the corresponding positions in the graphs. The locations of GC-rich expansion segments are highlighted by light blue rectangles. RNase L cleavage sites are highlighted in red.

Article Snippet: When RNAs from the PV-infected cells were analyzed by agarose gel electrophoresis, the accumulation of viral RNA was evident at 4–8 h post adsorption (hpa) ( B). rRNA fragments characteristic of RNase L activity were evident in RNAs from PV-infected W12 HeLa cells at 6 and 8 hpa, but these rRNA fragments were not detected in PV-infected M25 HeLa cells ( B, asterisks indicate the location of rRNA fragments characteristic of RNase L activity). cDNA libraries were prepared and sequenced using the RNAs from HeLa cells ( C and D and Supplementary Table S3 ). cDNA reads corresponding to PV RNA increased from undetectable levels at early times after infection to 5% of the cDNA at 6 hpa in W12 HeLa cells and 26.1% of the cDNA at 6 hpa in M25 HeLa cells ( C and D and Supplementary Table S3 ).

Techniques: Infection, Sequencing, Isolation

Journal: Nucleic Acids Research

Article Title: Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs

doi: 10.1093/nar/gku118

Figure Lengend Snippet: Host and viral RNA from mock-infected and PV-infected HeLa cells. RNA was isolated from mock-infected and PV-infected HeLa cells for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. ( A ) PV infection. W12 and M25 HeLa cells were infected with PV using 10 PFUs per cell. PV titers determined by plaque assay and plotted versus time (hpa). ( B ) RNA from PV-infected HeLa cells. RNA was isolated from infected cells, fractionated by agarose gel electrophoresis and visualized using ethidium bromide and UV light. ( C and D ) cDNA reads from W12 (C) and M25 (D) HeLa cells. The RNAs shown in Figure 3 B were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. Amounts of host and viral cDNA in each sample are plotted (data from Supplementary Table S3 ).

Article Snippet: When RNAs from the PV-infected cells were analyzed by agarose gel electrophoresis, the accumulation of viral RNA was evident at 4–8 h post adsorption (hpa) ( B). rRNA fragments characteristic of RNase L activity were evident in RNAs from PV-infected W12 HeLa cells at 6 and 8 hpa, but these rRNA fragments were not detected in PV-infected M25 HeLa cells ( B, asterisks indicate the location of rRNA fragments characteristic of RNase L activity). cDNA libraries were prepared and sequenced using the RNAs from HeLa cells ( C and D and Supplementary Table S3 ). cDNA reads corresponding to PV RNA increased from undetectable levels at early times after infection to 5% of the cDNA at 6 hpa in W12 HeLa cells and 26.1% of the cDNA at 6 hpa in M25 HeLa cells ( C and D and Supplementary Table S3 ).

Techniques: Infection, Isolation, Sequencing, Plaque Assay, Agarose Gel Electrophoresis

Journal: PLoS Genetics

Article Title: Transcriptome-Wide Mapping of 5-methylcytidine RNA Modifications in Bacteria, Archaea, and Yeast Reveals m5C within Archaeal mRNAs

doi: 10.1371/journal.pgen.1003602

Figure Lengend Snippet: Flow of the bisulfite-treatment/RNA-seq experiment. (A) Shown is a schematic representation of RNA molecules; colored boxes represent single bases. Red, blue, green and black represent C, U/T, A, and G residues, respectively. m 5 C modified cytosine bases are marked by a black circle. Total RNA is bisulfite-treated, leading to deamination of ‘C’ residues into ‘U’, except for methylated residues. Bisulfite-treated RNA is reverse transcribed into cDNA, which is then sequenced via an Illumina HiSeq system. This yields short sequence reads, representing random fragments of the sequenced RNAs. Resulting reads are mapped to the genome using an algorithm that allows mapping of ‘T’ residues in the sequenced cDNAs onto genomic ‘C's. Residues in which ‘C's are found to be consistently non-modified are declared as m 5 C (red arrow). (B) Flowchart of data analysis and artifact filtering ( Methods ).

Article Snippet: The mixed bisulfite-treated RNA sample was used as a template for cDNA library preparation according to the mRNA-seq Illumina protocol, omitting the polyA-based mRNA purification step.

Techniques: Flow Cytometry, RNA Sequencing Assay, Modification, Methylation, Sequencing

Journal: PLoS Genetics

Article Title: Transcriptome-Wide Mapping of 5-methylcytidine RNA Modifications in Bacteria, Archaea, and Yeast Reveals m5C within Archaeal mRNAs

doi: 10.1371/journal.pgen.1003602

Figure Lengend Snippet: RNA immunoprecipitation with modification-specific antibodies. Shown is the coverage of Illumina-sequenced cDNA following RNA fragmentation, antibody pulldown, reverse transcription and sequencing. Black line, pulldown performed with an anti-5-methylcitosine (hm 5 C) antibody; green line, pulldown performed with an anti-5-hydroxy-methylcitosine antibody; red line, input RNA (no antibody applied). X-axis, position along the genome; Y-axis (right), read coverage of the sequenced anti-m 5 C library; Y-axis (left), fold enrichment of peaks related to median coverage along the gene. The coverage of the anti-hm 5 C and input libraries was normalized using the median of the anti-m 5 C library as a reference point. (A) The 23S gene of S. solfataricus . Peaks corresponding to positions 2121 and 2643 in the gene (875,473 and 875,995 relative to the S. solfataricus genome, respectively) are marked. Another peak, which did not come up in our bisulfite-based analysis, is observed around position 2760. (B) The 16S gene of S. solfataricus . A single peak corresponding to position 1369 in the gene (position 873,040 relative to the genome) is marked. (C–E) Antibody pulldown of m 5 C modifications in protein-coding genes from Table 3 .

Article Snippet: The mixed bisulfite-treated RNA sample was used as a template for cDNA library preparation according to the mRNA-seq Illumina protocol, omitting the polyA-based mRNA purification step.

Techniques: Immunoprecipitation, Modification, Sequencing

Journal: BMC Genomics

Article Title: Quantitation of next generation sequencing library preparation protocol efficiencies using droplet digital PCR assays - a systematic comparison of DNA library preparation kits for Illumina sequencing

doi: 10.1186/s12864-016-2757-4

Figure Lengend Snippet: Bar charts showing the overall DNA library preparation yields of the different tested kits in comparison with the initial DNA input (500 ng). Except where mentioned otherwise all libraries were prepared using the original Illumina Paired end adaptor (also named Sanger adaptors) [ 6 , 22 ]. After end repair and A-tailing, the DNA loss was estimated using the PhiX specific primers ( 1st column ). After adaptor ligation, both the DNA amount (PhiX primers, 2nd column ) and the adaptor ligation efficiency (adaptor specific primers, 3rd column ) were measured, except for the Accel kits for which we were not able to measure directly the adaptor ligation efficiency as the adaptor sequences were unknown and are marked by a start (NM for not measured). After PCR, both the total amount of DNA (PhiX primers, 4th column ) and the amount of DNA bearing P5 and P7 primers at their ends were measured (P5 P7 primers, last column )

Article Snippet: Discussion We compared the practicability, reproducibility and quality of the libraries and sequencing data produced using 9 different kits to prepare Illumina DNA libraries.

Techniques: Ligation, Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Gene expression profiling of the aging mouse cardiac myocytes

doi:

Figure Lengend Snippet: Representative northern blot analyses confirming changes in mRNA levels. Total cellular RNA samples (2 µg) from young and aged LVCs were examined by northern blot analysis. Genes to which the probes correspond are identified to the left of the autoradiograms. Y, young LVCs; A, aged LVCs. Probes were radioactively labeled differentially expressed cDNA fragments. The level of mRNA specific for beta actin did not change significantly with aging and was used as an internal control.

Article Snippet: LVCs isolated from male and female hearts were used for the isolation of total RNA as suggested by the manufacturer (Qiagen). cDNA libraries were prepared using the SMART cDNA Synthesis Kit (Clontech) as suggested by the manufacturer.

Techniques: Northern Blot, Labeling

Journal: Journal of Clinical Microbiology

Article Title: Targeted Enrichment for Pathogen Detection and Characterization in Three Felid Species

doi: 10.1128/JCM.01463-16

Figure Lengend Snippet: (A) Targeted genome capture results in the enrichment and depletion of pathogen and host nucleic acids, respectively. RT-PCR was performed pre- and postcapture for feline genomic DNA/cDNA (GAPDH) and individual pathogens pre- and postcapture. Two unenriched samples (*) demonstrated an increase of ≥1-fold in the relative abundance of the host GAPDH gene, compared to all of the enriched samples tested, in which GAPDH either decreased or remained approximately the same. One sample (#) was enriched with only half of the recommended capture probes but still demonstrated a depletion of host nucleic acids and an increase in pathogen nucleic acids. All pathogen nucleic acids measured increased in relative abundance postenrichment. Pathogen and host nucleic acids were measured only in samples with sufficient volume remaining before and after sequencing to conduct RT-PCRs. (B) Pathogen nucleic acids were enriched from 58-fold to 56 million-fold relative to host nucleic acids. The relative abundance of pathogen DNA did not increase in the two unenriched samples (*), but the corresponding paired, enriched pathogens increased over 1 million-fold. Samples in this figure are grouped by pathogen type: FFV, feline foamy virus; FIVA, feline immunodeficiency virus clade A; FIVC, feline immunodeficiency virus clade C; PLVB, puma lentivirus clade B; FeLV, feline leukemia virus; FCoV, feline corona virus; FCV, feline calicivirus.

Article Snippet: Each DNA/cDNA library was barcoded via ligation of standard 6-mer oligonucleotides (Illumina, Inc., San Diego, CA) for multiplex sequencing.

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing