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To perform a standard RNAseq experiment to evaluate the impact of the knockout of Zeus gene(CG9573) in D. melanogaster

ABSTRACT: This experiment was designed to study the transcriptional alternations using RNA-seq technique. RNA materials were collected and sequenced with multiple experimental groups in tri-plicates as follows: control-knockout-replicate 1~3 (KO-Ctrl__1~3), Zeus-knockout-replicate 1~3 (KO-Zeus__1~3), for RNA-sequencing. In total, 6 sequencing files were downloaded from the genomic core facility, University of Chicago. The aims of this analysis are to 1) conduct quality control (QC) assessment on the original raw sequencing data, 2) map sequencing reads to target species, 3) identify differentially expressed (DE) protein-coding genes per comparison. Through assessing the quality of original raw sequencing data, good base-wise quality was revealed in average in all samples, indicating a high confidence on the base call. Adapter/primer sequences are absent from reads; therefore, trimming of sequencing reads was skipped prior to downstream RNA-seq analysis. Overall, around 94.89% of reads were successfully mapped to the fruit fly genome model in RNA-seq data. All RNA samples were used for further downstream identification of differentially expressed (DE) mRNA genes in individual pairwise comparison. Several commonly used tools (DESeq2, edgeR, and limma) were utilized for the identification of DE mRNA between pairwise groups. Using the criteria of fold change no less than 1.5 and FDR (False Discovery Rate) less than 0.1). Pairwise comparison, KO-Zeus/KO-Ctrl, were conducted. Overall, we find Zeus regulated the expression of 661 differentially expressed genes (DEGs) based on combination of three tools. Venn diagram was further depicted to present the consensus/overlapped DE gene sets detected across different methods.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE192879 | GEO | 2022/01/04

REPOSITORIES: GEO

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Project description:We have established a certification system for antibodies to be used in chromatin immunoprecipitation assays coupled to massive parallel sequencing (ChIP-seq). This certification comprises a standardized ChIP procedure and the attribution of a numerical quality control indicator (QCi) to biological replicate experiments. The QCi computation is based on a universally applicable quality assessment that quantitates the global deviation of randomly sampled subsets of ChIP-seq dataset with the original genome-aligned sequence reads. Comparison with a QCi database for >28,000 ChIP-seq assays were used to attribute quality grades (ranging from âAAAâ to âDDDâ) to a given dataset. In the present report we used the numerical QC system to assess the factors influencing the quality of ChIP-seq assays, including the nature of the target, the sequencing depth and the commercial source of the antibody. We have used this approach specifically to certify mono and polyclonal antibodies obtained from Active Motif directed against the histone modification marks H3K4me3, H3K27ac and H3K9ac for ChIP-seq. The antibodies received the grades AAA to BBA (www.ngs-qc.org). We propose to attribute such quantitative grading of all antibodies attributed with the label âChIP-seq gradeâ. The NGS-QC team has setup a certification procedure for Antibodies dedicated to ChIP-seq applications based on (1)a highly reproducible pipeline for performing ChIP-seq assays including the use of an automated liquid handling platform (TECAN EVO75) for ChIP and sequencing library preparation steps and (2) the use of the NGS-QC Generator tool for providing a ChIP-seq quality grade and to evaluate other parameters like the Optimal sequencing depth, the degree of reproducibility among biological replicates and the comparison of the quality grades of the certified antibody with datasets retrieved in the public domain. This quality certification structure makes possible to assign a measured Quality Stamp to ChIP-seq grade Antibodies.

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To perform a standard RNAseq experiment to evaluate the impact of the knockout of Zeus gene(CG9573) in D. melanogaster

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