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: We exploited the 2′, 3′-cyclic phosphates at RNA cleavage sites to make cDNA libraries suitable for Illumina sequencing ( Supplementary Figure S1 ).

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: We exploited the 2′, 3′-cyclic phosphates at RNA cleavage sites to make cDNA libraries suitable for Illumina sequencing ( Supplementary Figure S1 ).

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: We exploited the 2′, 3′-cyclic phosphates at RNA cleavage sites to make cDNA libraries suitable for Illumina sequencing ( Supplementary Figure S1 ).

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: We exploited the 2′, 3′-cyclic phosphates at RNA cleavage sites to make cDNA libraries suitable for Illumina sequencing ( Supplementary Figure S1 ).

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: We exploited the 2′, 3′-cyclic phosphates at RNA cleavage sites to make cDNA libraries suitable for Illumina sequencing ( Supplementary Figure S1 ).

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: We exploited the 2′, 3′-cyclic phosphates at RNA cleavage sites to make cDNA libraries suitable for Illumina sequencing ( Supplementary Figure S1 ).

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

Journal: PLoS ONE

Article Title: Role of Global and Local Topology in the Regulation of Gene Expression in Streptococcus pneumoniae

doi: 10.1371/journal.pone.0101574

Figure Lengend Snippet: Domains show conservation and expression tendencies. (A) Relationship between gene frequency and AT content in microarrays hybridized with cDNAs obtained from total RNA. (B) Relationship between average FU and AT content in microarrays hybridized with cDNAs obtained from total RNA. A window of 11 genes (average value on the middle one) was applied for FU values. Only coding regions were considered in the AT calculations. (C) Relationship between FU value and AT content in microarrays hybridized with cDNA obtained from total DNA, the median of about 32 microarray spot values was calculated and the mean of medians from two replicates considered. (D) Relationship between the gene lack index and FU. Genes were considered equivalent to R6 counterparts when their polypeptide products shared ≥80% identity over ≥80% of the sequence length in other S. pneumoniae strains with complete genome sequence: 70585, ATCC 700669, CGSP14, D39, G54, Hungary19A-6, JJA, P1031, Taiwan19F-14, TCH8431/19A and TIGR4. An 11-gene window (roughly equivalent to 10 Kb) was applied. Misleading continuum of sharp peaks or poorly defined peaks were obtained when narrower or wider windows were applied, respectively.

Article Snippet: RNA techniques Synthesis of cDNAs from 5 µg of total RNA was performed as previously described . cDNAs obtained were subjected to quantitative qRT-PCR (Chromo 4, BioRad) in 20 µl reactions containing 2 µl of cDNA, 0.4 µM of each specific primer, and 10 µl of LightCycler FastStart Universal A SYBR Green Master (Roche).

Techniques: Expressing, Microarray, Sequencing