cap analysis gene expression (cage) library preparation, sequencing, mapping, and gene expression analysis  (DNAFORM Inc)

Journal: bioRxiv

Article Title: Novel Herpesvirus Transcripts with Putative Regulatory Roles in DNA Replication and Global Transcription

doi: 10.1101/2023.03.25.534217

Figure Lengend Snippet: Techniques applied for the generation of novel sequencing data, including infection of the cells with various viruses, library preparation, sequencing and bioinformatics. The workflow of the qPCR validations of several transcripts is also illustrated. Abbreviations: PA: polyadenylated RNAs; T: Terminator-handled samples; RD: ribodepleted RNAs; dcDNA: direct cDNA-seq; CAGE: Cap Analysis Gene Expression-Seq; dRNA: direct RNA-Seq; qRNA: short-read RNA-Seq (library generated by qRNA-seq kit); acDNA: amplified cDNA-Seq.

Article Snippet: Additionally, Cap Analysis of Gene Expression (CAGE) sequencing (CAGE-Seq, running on Illumina platform) was used for defining the TSS landscape in EHV-1 and KSHV ( ).

Techniques: Sequencing, Infection, Expressing, RNA Sequencing Assay, Generated, Amplification

Journal: bioRxiv

Article Title: Novel Herpesvirus Transcripts with Putative Regulatory Roles in DNA Replication and Global Transcription

doi: 10.1101/2023.03.25.534217

Figure Lengend Snippet: TSS distributions are illustrated in the following genomic regions of EHV-1 and KSHV: A. EHV-1 OriL; B. EHV-1 OriS of the IRS; C. KSHV OriLyt-R; D. KSHV OriLyt-L; E. KSHV OriLyt-L – here a higher resolution is used for the better visibility of the low-abundance TSSs. It can be seen that CTO-S transcript is at a high level (A), whereas NOIR-1 is a group of transcripts expressed in a relative low level. Smoothed density plots of the 5′ ends in the CAGE data. Here, y-axis shows the result of the probability estimation of the 5′ ends using a probability density function (details are described in the Materials and Methods section). Coding sequence (CDS) annotations for the respective genomes (shown with the accession number at the right) were visualized in the lower part. The coverage of the positive strand and the CDS annotation are shown in red while these were illustrated in blue in the negative strand. The Ori regions in EHV-1 are illustrated in black whereas in KSHV they are depicted in green – in the latter case, an accompanying white box shows the 20-nt binding site for the DNA replication origin-binding protein.

Article Snippet: Additionally, Cap Analysis of Gene Expression (CAGE) sequencing (CAGE-Seq, running on Illumina platform) was used for defining the TSS landscape in EHV-1 and KSHV ( ).

Techniques: Sequencing, Binding Assay

Journal: eLife

Article Title: Polysome-CAGE of TCL1-driven chronic lymphocytic leukemia revealed multiple N-terminally altered epigenetic regulators and a translation stress signature

doi: 10.7554/eLife.77714

Figure Lengend Snippet: ( A ) A scheme of the Cap Analysis Gene Expression (CAGE) in healthy (WT) and CLL B cells (Eµ-Tcl1). WT and Eµ-Tcl1 splenic B cells were isolated using CD5 and CD19 magnetic beads. Total RNA samples were subjected to the CAGE as described in Materials and methods. CAGE-derived TSSs (CTSSs) were mapped and clustered into genomic blocks referred as tag clusters (blue bar) with a peak of most recovered CTSS (blue tick) indicates the TSSs in a single-base resolution. ( B ) Metagene analysis of Eµ-Tcl1 (orange) and WT (gray) CAGE libraries aligned to annotated TSS atlas of the FANTOM5 project. ( C ) The frequency (left) and expression level (right, log2 TPM) of CAGE tag clusters in Eµ-Tcl1 (orange) and WT (gray) mice by gene locations. The percentage values on the columns of intronic and intergenic locations refer to predicted enhancer RNA (eRNA) TSSs. ( D ) Differentially expressed (DE) TSSs. Left circle indicates unchanged (light gray) and significantly DE (brown) TSS between Eµ-Tcl1 and WT mice. Right circle details frequencies of Eµ-Tcl1 upregulated genic and eRNA TSSs (orange and dark orange, respectively) and downregulated genic and eRNA TSSs (gray and dark gray). Subgrouping of DE TSSs that are positively correlated with enhancers are indicated. ( E ) Sequence LOGOs (−40 to +30 relative to the TSS) of Eµ-Tcl1 upregulated (lower panel) and downregulated (upper panel) sharp promoters where TATA box situated between −31 and −24 sites and cap-proximal region (12 bases) are boxed in a dashed line.

Article Snippet: Five μg RNA samples were subjected to library preparation of Cap Analysis Gene Expression CAGE using CAGE protocol adapted for Illumina sequencing ( ).

Techniques: Gene Expression, Isolation, Magnetic Beads, Derivative Assay, Expressing, Sequencing

Journal: eLife

Article Title: Polysome-CAGE of TCL1-driven chronic lymphocytic leukemia revealed multiple N-terminally altered epigenetic regulators and a translation stress signature

doi: 10.7554/eLife.77714

Figure Lengend Snippet: ( A ) Coefficient scores of Cap Analysis of Gene Expression (CAGE) library replicates of Eu-Tcl1 and WT total RNA samples calculated using Pearson pairwise-correlation test. ( B ) Genome browser example of an enhancer candidate showing balanced bidirectional transcription of enhancer RNAs (eRNAs). Tag-Per-Million per values plotted in blue for plus strand and in red for minus strand. ( C ) An example of a super enhancer. Closely spaced enhancers positioned within 18,405 bps range. ( D ) ‘Blind’ version of the variance-stabilizing transformation of the two replicates of Eu-Tcl1 and WT CAGE samples. ( E ) Bimodal distribution of the widths of tag clusters (TCs) of highly expressed transcription start sites (TSSs). Most CAGE TCs are distributed either below (Sharp) or above (Broad) 10 bases distance holding 10–90% of pooled CAGE tags. ( F ) Sequence logos of core promoter regions (−40 and +30 bp) of Sharp and Broad classes of TCs.

Article Snippet: Five μg RNA samples were subjected to library preparation of Cap Analysis Gene Expression CAGE using CAGE protocol adapted for Illumina sequencing ( ).

Techniques: Gene Expression, Transformation Assay, Sequencing

Journal: eLife

Article Title: Polysome-CAGE of TCL1-driven chronic lymphocytic leukemia revealed multiple N-terminally altered epigenetic regulators and a translation stress signature

doi: 10.7554/eLife.77714

Figure Lengend Snippet: The upper panel shows a scheme of the 5′ RACE followed by qPCR (see Materials and methods for details). The lower panel shows the qPCR results of the indicated chromatin modifiers. The total RNA samples of Eu-Tcl1 and WT mice are the same ones used for the CAGE.

Article Snippet: Five μg RNA samples were subjected to library preparation of Cap Analysis Gene Expression CAGE using CAGE protocol adapted for Illumina sequencing ( ).

Techniques:

Journal: eLife

Article Title: Polysome-CAGE of TCL1-driven chronic lymphocytic leukemia revealed multiple N-terminally altered epigenetic regulators and a translation stress signature

doi: 10.7554/eLife.77714

Figure Lengend Snippet: ( A ) Coefficient scores of Cap Analysis of Gene Expression (CAGE) library replicates of Eu-Tcl1 polysome-free (Free) and polysomes RNA samples calculated using Pearson pairwise-correlation test. ( B ) Frequency and boxplot of expression level (log2(TPM)) of CAGE tag clusters (TCs) in polysome-free (light blue) and polysomes (dark blue) by gene-structure locations. ( C ) Boxplot of expression level (log2(TPM)) of TCs classified as transcription start site (TSS) or enhancer in polysome-free (light blue) and Heavy polysomes (dark blue). ( D ) Example of differentially translated isoforms originated by alternative promoters (P1 and P2), differing in 5′ UTR lengths. TSS coverage tracks of polysome-free (Free) and Heavy + Light polysomes are used for calculating TE values of the alternative isoforms. When the TE of the one isoform (shorter 5′ UTR, AP-2) is 2FC greater than the longer isoform (AP-1), the first assigned as Low-TE isoform and the latter as High-TE isoform.

Article Snippet: Five μg RNA samples were subjected to library preparation of Cap Analysis Gene Expression CAGE using CAGE protocol adapted for Illumina sequencing ( ).

Techniques: Gene Expression, Expressing