Journal: bioRxiv

Article Title: Genome wide CRISPR screen for Pasteurella multocida toxin (PMT) binding proteins reveals LDL Receptor Related Protein1 (LRP1) as crucial cellular receptor

doi: 10.1101/2022.08.04.502755

Figure Lengend Snippet: Mouse embryonic fibroblasts (MEF) stably expressing Flag-Cas9-EGFP were transduced with a lentiviral CRISPR-library. Cells were treated three times with PMT(C1165S)DTa and surviving cells grown. Genomic DNA was extracted, inserted sgRNA amplified and sequenced.

Article Snippet: We utilized a well-functioning and validate genome-wide mouse lentiviral CRISPR guide RNA library v2 (Addgene).

Techniques: Stable Transfection, Expressing, Transduction, CRISPR, Amplification

Journal: bioRxiv

Article Title: Genome wide CRISPR screen for Pasteurella multocida toxin (PMT) binding proteins reveals LDL Receptor Related Protein1 (LRP1) as crucial cellular receptor

doi: 10.1101/2022.08.04.502755

Figure Lengend Snippet: The volcano plot shows gene expression changes between cells treated with lethal PMT-DTa chimera and those only transduced with the CRISPR library. All reads were mapped locally using BWA-MEM [ ,6], then quantified with featureCounts [4], and finally fold changes between the condition were calculated by DESeq2 [7] (see galaxy history). The volcano plot was drawn with the bioinfokit toolkit [8]. Significantly enriched genes, having a positive fold change above 0.584 and a p-value lower or equal 5%, are shown in blue. The significance thresholds are marked by gray-dotted lines. The ten most significant genes are highlighted with their name. Non-significant genes are colored in gray.

Article Snippet: We utilized a well-functioning and validate genome-wide mouse lentiviral CRISPR guide RNA library v2 (Addgene).

Techniques: Gene Expression, Transduction, CRISPR

Journal: Frontiers in Genetics

Article Title: Addressing Bias in Small RNA Library Preparation for Sequencing: A New Protocol Recovers MicroRNAs that Evade Capture by Current Methods

doi: 10.3389/fgene.2015.00352

Figure Lengend Snippet: Comparison of miRNA expression profiles among different library preparation protocols reveals massive differential bias. A comparison of the following four methods is shown: Illumina v1.5 library preparation sequenced on GAIIx platform (v1.5_GAIIx), Illumina TruSeq library preparation sequenced on GAIIx platform (TS_GAIIx), Illumina TruSeq library preparation sequenced on HiSeq platform (TS_HiSeq) and Bioo Scientific NEXTflexV2 library preparation sequenced on the HiSeq platform (NF-HiSeq). Three biological replicate small RNA libraries were generated for each of the first three methods and one replicate was generated for the NF-HiSeq method. (A) Correlation of miRNA profiles between each pair of datasets (correlation values were calculated by Pearson’s metric). Similar results were obtained with Spearman’s correlation coefficient, rho (data not shown). White and blue colors indicate strongest and weakest correlation, respectively. (B) miRNA expression profiles across all 10 samples. Hierarchical clustering was used to identify samples with closely related expression profiles. Expression is represented as z -score, indicating the number of standard deviations below (purple) or above (orange) the mean across all ten libraries. Both (A,B) used only the set of miRNAs identified as “highly expressed” ( n = 358).

Article Snippet: We isolated RNA from a widely used pancreatic beta-cell-like cell line (MIN6) and performed small RNA-seq using four different methods: (1) Illumina v1.5 library preparation sequenced on GAIIx platform (v1.5-GAIIx), (2) Illumina TruSeq library preparation sequenced on GAIIx platform (TS-GAIIx), (3) Illumina TruSeq library preparation sequenced on HiSeq platform (TS-HiSeq), and (4) Bioo Scientific NEXTflex V2 library preparation sequenced on the HiSeq platform (NF-HiSeq).

Techniques: Comparison, Expressing, Generated

Journal: Frontiers in Genetics

Article Title: Addressing Bias in Small RNA Library Preparation for Sequencing: A New Protocol Recovers MicroRNAs that Evade Capture by Current Methods

doi: 10.3389/fgene.2015.00352

Figure Lengend Snippet: Fifty of the most abundant miRNAs are greater than ten-fold differentially detected between Illumina v1.5 and TruSeq. (A) Comparison of relative expression levels of miRNAs in MIN6 ( n = 358) between the GAIIx and HiSeq sequencing platforms with libraries prepared by TruSeq (TS) is shown. Each data point represents the average relative expression level for an individual miRNA across three biological replicates. (B) Comparison of relative expression levels of miRNAs in MIN6 ( n = 358) between the v1.5 and TruSeq (TS) library preparation methods is shown. Each data point represents the average relative expression level for an individual miRNA across three biological replicates. (C,D) Comparison of relative expression levels of miRNAs in MIN6 ( n = 358, C ) and mouse liver ( n = 178, D ) between the TruSeq (TS) and NEXTflex (NF) library preparation methods is shown. Each data point represents the average relative expression level for an individual miRNA across three biological replicates (A,B) , or one biological replicate (C,D) . Relative miRNA expression levels were calculated according to the following: log 10 (mean(miRNA RPMM)), where RPMM is reads per million mapped reads. Pearson correlation values are displayed in red text within each panel, and gray dashed lines denote 10-fold differential expression.

Article Snippet: We isolated RNA from a widely used pancreatic beta-cell-like cell line (MIN6) and performed small RNA-seq using four different methods: (1) Illumina v1.5 library preparation sequenced on GAIIx platform (v1.5-GAIIx), (2) Illumina TruSeq library preparation sequenced on GAIIx platform (TS-GAIIx), (3) Illumina TruSeq library preparation sequenced on HiSeq platform (TS-HiSeq), and (4) Bioo Scientific NEXTflex V2 library preparation sequenced on the HiSeq platform (NF-HiSeq).

Techniques: Comparison, Expressing, Sequencing, Quantitative Proteomics