Journal: Nucleic Acids Research

Article Title: Grouped-seq for integrated phenotypic and transcriptomic screening of patient-derived tumor organoids

doi: 10.1093/nar/gkab1201

Figure Lengend Snippet: Performance characterization of the Grouped-seq platform. ( A ) Mapped read and gene numbers across 192 microwells of the SMARchip. ( B ) Heatmap of correlation coefficients between any two of microwells. ( C ) Top 15 genes with the highest expression levels in all the microwells. ( D ) Quantitative capability of the Grouped-seq evaluated by ERCC sample. ( E ) Mixed-species experiment with human (A549) and mouse (3T3) cells performed on a single SMARchip. ( F ) High correlation between the Grouped-seq and the off-chip TruSeq. ( G ) Read and gene numbers of 192 microwells obtained at different sequencing depth from 0.025, 0.05, 0.1, 0.5, to 1 M reads per microwell. ( H ) UMI correction rates at different sequencing depth. UMI correction rate is defined as the ratio of read number to UMI number. ( I ) Limit of detection (LOD) of the gene expression level that can be detected at the different sequencing depths. The counts per million (CPMs) of the genes obtained at the 1-M sequencing depth are used as the standards and the LOD of a sequencing depth is defined as the upper limit of the CPMs (obtained at 1-M sequencing depth) of the genes that can be detected at least once with this sequencing depth.

Article Snippet: Meantime, 10 5 of A549 cells from the same batch were collected to extract total RNA using a RNeasy column (Qiagen) and a sequencing library was prepared from the total RNA using the Illumina TruSeq poly(A) + RNA-Seq library construction according to the manufacturer's instruction.

Techniques: Expressing, Sequencing

Journal: Nature Communications

Article Title: Long non-coding RNA Gm15441 attenuates hepatic inflammasome activation in response to PPARA agonism and fasting

doi: 10.1038/s41467-020-19554-7

Figure Lengend Snippet: a Venn diagrams of RefSeq transcripts and lncRNA transcripts that were differentially regulated by WY-14643 (fold change > 2 at FDR < 0.05) in wild-type and in Ppara −/− mouse liver, as determined by RNA-seq. 123 of the RefSeq genes are non-coding, indicated by their NR accession numbers. Six RefSeq genes and six lncRNA genes show opposite responses to WY-14643 treatment and are excluded from the gene counts shown. b Relative basal lncRNA expression in select tissues. c LncRNA expression in tissues from Ppara +/+ and Ppara −/− mice treated with WY-14643 for 48 h. At least five mice were analyzed for each genotype and treatment group. Each bar represents the mean ± SD for n = 5 tissue samples. Adjusted p values, provided in the panels, as determined by ANOVA with Tukey’s multiple comparison correction, one-sided for comparisons to Ppara +/+ mice or as shown (dashed horizontal lines). ND not detected.

Article Snippet: High quality RNA samples (RIN > 9.0) were pooled, as described below, and used to construct stranded RNA-seq libraries from polyA-selected total liver RNA using an Illumina stranded TruSeq mRNA Prep Kit (Illumina, San Diego, CA, USA).

Techniques: RNA Sequencing, Expressing, Comparison

Journal: Nature Communications

Article Title: Long non-coding RNA Gm15441 attenuates hepatic inflammasome activation in response to PPARA agonism and fasting

doi: 10.1038/s41467-020-19554-7

Figure Lengend Snippet: Wild-type ( Ppara +/+ ) mice were treated with WY-14643 and stranded RNA-seq was performed on total liver mRNA. a Expression of the antisense lncRNA Gm15441 is strongly upregulated. b Expression of the protein-coding Txnip mRNA is downregulated. Shown are the changes in expression of Gm15441 ( c ), and Txnip ( d ), mRNAs in Ppara +/+ and Ppara −/− mice treated with WY-14643, or vehicle control, for 48 h, determined by qRT-PCR. e Time course for changes in expression of Gm15441 and Txnip mRNA over a 24 h period following treatment with WY-14643 by gavage, determined by qRT-PCR. The maximum response of Txnip mRNA was seen at 1.5 h and for Gm15441 was seen at 6 h. Each data point represents the mean ± SD for n = 5 liver samples. Adjusted p values, provided in the panels, as determined by ANOVA with Tukey’s multiple comparison correction, two-sided for comparisons to Ppara +/+ mice or as shown (dashed horizontal line).

Article Snippet: High quality RNA samples (RIN > 9.0) were pooled, as described below, and used to construct stranded RNA-seq libraries from polyA-selected total liver RNA using an Illumina stranded TruSeq mRNA Prep Kit (Illumina, San Diego, CA, USA).

Techniques: RNA Sequencing, Expressing, Control, Quantitative RT-PCR, Comparison

Journal: Nature Communications

Article Title: Long non-coding RNA Gm15441 attenuates hepatic inflammasome activation in response to PPARA agonism and fasting

doi: 10.1038/s41467-020-19554-7

Figure Lengend Snippet: a Schematic representation of GFP construct inserts with or without the TXNIP 5′UTR sequence. b Analysis of Gm15441 RNA and Gfp mRNA in Hepa-1 cells. c Western blot analysis of GFP protein and relative density of GFP signal from Hepa-1 cells (right). d Fluorescence of GFP in Hepa-1 cells transfected with GFP with empty or Gm15441 plasmid DNA for 48 h ( n = 3). Scale bars represent 20 nm (100x). e Fluorescence of GFP in Hepa-1 cells transfected with 5′ UTR sequence containing GFP with empty or Gm15441 plasmid DNA for 48 h ( n = 3). Scale bars represent 20 nm (100x). f Analysis of Hfe2 , Pol3gl , and Ankrd34a mRNAs from Hepa-1 and NIHT3T cells transfected with empty plasmid or Gm15441 expression vector for 24 h. Each data point represents mean ± SD for n = 3 replicates. Adjusted p values, provided in the panels, as determined by ANOVA with Tukey’s multiple comparison correction, one-sided for comparisons in the absence of Gm15441 ( b , c ) or to empty vector ( f ). g Model for role of Gm15441 in suppressing TXNIP-mediated inflammasome activation.

Article Snippet: High quality RNA samples (RIN > 9.0) were pooled, as described below, and used to construct stranded RNA-seq libraries from polyA-selected total liver RNA using an Illumina stranded TruSeq mRNA Prep Kit (Illumina, San Diego, CA, USA).

Techniques: Construct, Sequencing, Western Blot, Fluorescence, Transfection, Plasmid Preparation, Expressing, Comparison, Activation Assay