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RNA sequencing profiles of control and PRPF19 knockdown TIG-3 cells

ABSTRACT: Total RNA was purified from TIG-3 cells transfected with siControl or siPRPF19 using miRNeasy Mini Kit (QIAGEN) with RNase-free DNase (QIAGEN). Library preparation, sequencing, and data analysis were performed by DNAFORM (Yokohama, Kanagawa, Japan). Quality and quantity of extracted RNA was assessed by NanoDrop 8000 Microvolume UV-Vis spectrophotometer (Thermo Fisher Scientific) and Agilent RNA 6000 Nano kit of BioAnalyzer 2100 System (Agilent Technologies). RNA-seq library was prepared using SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian (Takara Bio) following the manufacturer’s instruction. In brief, ribosomal RNA was depleted employed RiboGone which included in the kit. After that, first strand cDNA synthesis was performed using N6 primer. The first-stranded cDNA was purified using equal volume of AMPure XP beads (Beckman Coulter). The purified cDNA was amplified into Illumina-specific RNA-seq libraries by PCR. The libraries were sequenced on a Hiseq sequencer (Illumina) to generate 150 nt paired-end reads. The quality of sequence data was first assessed using FastQC (ver. 0.11.7). Raw sequence reads were then trimmed and quality-filtered with Trim Galore! (ver. 0.4.4), Trimmomatic (ver. 0.36), and cutadapt (ver. 1.16) software. Trimmed reads were mapped to the human reference genome (GRCh38.p10) using STAR (ver. 2.6.1a).

ORGANISM(S): Homo sapiens

PROVIDER: GSE168391 | GEO | 2021/07/20

REPOSITORIES: GEO

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Project description:Purpose: We identified novel methyl reader domain protein in P. falciparum. Using Next Generation Sequencing, we studied the differential gene expression in PfCDP conditionally deleted P. falciparum versus wild type. Methods: Transcriptome analysis of PfCDP KO vs WT P. falciparum using high throughput RNA sequencing in triplicate (Three controls and three KO samples). The total RNA was isolated from P. falciparum WT and PfCDP KO lines, the quality of RNA was assessed using Nanodrop2000, and Bioanalyzer 2100. RNA sequencing libraries were prepared with Illumina-compatible NEBNext® Ultra™ II Directional RNA Library Prep Kit. The double stranded cDNA was purified using JetSeq Beads and the purified cDNA was end-repaired, adenylated and ligated to Illumina multiplex barcode adapters as per NEBNext® Ultra™ II Directional RNA Library Prep protocol. The libraries were sequenced on IlluminaHiSeq 4000 sequencer for 150 bp paired-end chemistry. The data obtained from the sequencing run was de-multiplexed using Bcl2fastq software v2.20 and FastQ files were generated based on the unique dual barcode sequences. The sequencing quality was assessed using the FastQC software ver. 0.11.9. The adaptor sequences and low quality bases (phred quality cutoff set at 30) were trimmed from the reads using the Trim Galore tool ver. 0.6.5. The reads that survived quality check were aligned to the Plasmodium falciparum 3D7 isolate genome (ver. 46) using the Hisat2 read alignment tool. The library strandedness was set as firststrand in accordance with the strand specific library preparation protocol. The alignement output files in the sam format were sorted and converted to the bam format using the Samtools utilities (ver 1.10). Results: The transcript abundance count data was generated using the HTSeq-count function (ver. 0.12.4). Differential gene expression analysis was performed using the DESeq2 package (ver. 3.11) in R programming environment. Genes were classified as deregulated on the basis of log2fold-change cutoff of +1 (for upregulation) and -1 (for downregulation) and p-value cutoff of 0.05. Gene Ontology analysis was performed using the GO tool hosted by the PlasmoDB web database. Data visualisation of the DGE was performed using custom scripts in R programming environment and with DESeq2 package utilities. The DGE analysis has revealed that up regulation of a subsets virulence genes family with significant p values. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

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RNA sequencing profiles of control and PRPF19 knockdown TIG-3 cells

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