Journal: BMC Genomics

Article Title: Single nucleotide resolution RNA-seq uncovers new regulatory mechanisms in the opportunistic pathogen Streptococcus agalactiae

doi: 10.1186/s12864-015-1583-4

Figure Lengend Snippet: Characterization of transcription start sites in S. agalactiae. A . Visualization of sequence reads mapped to the genome of strain NEM316 in conditions of dRNA-seq: strand-specific sequencing of transcript 5′ ends with (TAP+) and without (TAP-) TAP treatment, and strand-specific RNA-seq. Two dRNA-seq experiments are shown corresponding to 1: RNA from multiple growth conditions (MG sample); 2: RNA from a ∆covRS mutant grown to mid-exponential phase. The RNA-seq library was prepared with the wt strain at mid-exponential phase. B . Detailed view of the 958000–978000 region. Protein coding genes annotated on the (+) and (−) strands are indicated by red and blue large arrows. TSSs are depicted as small arrows. Based on dRNA-seq and RNA-seq data, a transcript corresponding to a ncRNA (srn040/tmRNA) was annotated and is shown as a large green arrow . C . Proportions of the total TSSs detected under each experimental condition. Grey: TSSs detected with RNA from multiple growth conditions (wt, MG); red: TSSs not detected in wt, MG. MG: mixture of growth conditions; LE: late exponential phase; midE: mid-exponential growth phase; AcStr: acid stress. D : Proportions of TSSs according to the number of experiments in which they were detected.

Article Snippet: RNA ligated to the two adapters was reverse-transcribed with the Superscript II Reverse Transcriptase (Life Technologies) and the RNA reverse transcription primer (Illumina TruSeq Small RNA kit).

Techniques: Sequencing, RNA Sequencing Assay, Mutagenesis

Journal: BMC Genomics

Article Title: Single nucleotide resolution RNA-seq uncovers new regulatory mechanisms in the opportunistic pathogen Streptococcus agalactiae

doi: 10.1186/s12864-015-1583-4

Figure Lengend Snippet: Identification of a novel riboswitch upstream gbs1262. A . Transcriptional organization deduced from dRNA-seq and RNA-seq experiments. Reads aligning upstream gbs1262 were visualized by IGV browser . The sequence of the sRNA resulting from transcription premature arrest at a rho-independent terminator is indicated as a grey arrow. B . Secondary structure predicted by RNalifold , based on the alignment of 14 sequences similar to gbs1262 5′UTR in Lactobacillales and upstream a potential tryptophan-related gene in F. nucleatum (Additional file ). The two conserved folded structures are indicated in red and green boxes, with the green box corresponding to a rho-independent terminator. Sequence covariations supporting the consensus structure are marked by color: red marks pairs with no sequence variation; ochre and green mark pairs with 2 or 3 types of pairs, respectively. C and D . Alternative structures of gbs1262 putative riboswitch as determined by mfold , showing the formation of an antiterminator structure .

Article Snippet: RNA ligated to the two adapters was reverse-transcribed with the Superscript II Reverse Transcriptase (Life Technologies) and the RNA reverse transcription primer (Illumina TruSeq Small RNA kit).

Techniques: RNA Sequencing Assay, Sequencing

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Probing transcriptome-wide RNA structural changes dependent on the DEAD-box helicase Dbp2

doi: 10.1007/978-1-0716-0935-4_18

Figure Lengend Snippet:

Article Snippet: Set up a PCR reaction as below: table ft1 table-wrap mode="anchored" t5 Component Volume (μL) Final quantity Ligated cDNA (or water for primer-dimer control) 5 μL ? dNTP mixture, 2.5 mM (provided with Takara Ex Taq) 2 0.2 mM Ex Taq buffer, 10X 2.5 1X Illumina TruSeq forward primer (5 μM) 1 0.2 μM Illumina TruSeq reverse primer (5 μM) 1 0.2 μM TaKaRa Ex Taq (50 U/μL) 0.5 0.1 U/μL Nuclease-free water 13 13 μL Open in a separate window Amplify the cDNAs by PCR using the following conditions ( Note 9 ). table ft1 table-wrap mode="anchored" t5 Step Temperature Time Number of cycles Initial denaturation 98 °C 1 min 1 Denaturation 98 °C 10 sec 10 – 35 Annealing 55 °C 30 sec Extension 72 °C 1 min Final extension 72 °C 10 min 1 Incubation 4 °C 2 min 1 Open in a separate window Resolve PCR products by PAGE ( Note 10 ), and collect fragments larger than 150 bp as described in section 3.4 , step 8 – 10.

Techniques:

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Probing transcriptome-wide RNA structural changes dependent on the DEAD-box helicase Dbp2

doi: 10.1007/978-1-0716-0935-4_18

Figure Lengend Snippet:

Article Snippet: Set up a PCR reaction as below: table ft1 table-wrap mode="anchored" t5 Component Volume (μL) Final quantity Ligated cDNA (or water for primer-dimer control) 5 μL ? dNTP mixture, 2.5 mM (provided with Takara Ex Taq) 2 0.2 mM Ex Taq buffer, 10X 2.5 1X Illumina TruSeq forward primer (5 μM) 1 0.2 μM Illumina TruSeq reverse primer (5 μM) 1 0.2 μM TaKaRa Ex Taq (50 U/μL) 0.5 0.1 U/μL Nuclease-free water 13 13 μL Open in a separate window Amplify the cDNAs by PCR using the following conditions ( Note 9 ). table ft1 table-wrap mode="anchored" t5 Step Temperature Time Number of cycles Initial denaturation 98 °C 1 min 1 Denaturation 98 °C 10 sec 10 – 35 Annealing 55 °C 30 sec Extension 72 °C 1 min Final extension 72 °C 10 min 1 Incubation 4 °C 2 min 1 Open in a separate window Resolve PCR products by PAGE ( Note 10 ), and collect fragments larger than 150 bp as described in section 3.4 , step 8 – 10.

Techniques: