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SRP045421
Homo sapiens
24 Downloadable Samples
Illumina HiSeq 2000
Description
We extracted RNA from whole cells and RNA from the cytoplasm and performed RNA sequening to compare differences in gene expression level and investigate what is the most appropriate estimate of the amount of mRNA present in a given cell population. The study was based on three human cell lines. Overall design: Analyze of transcriptome in 3 human cell lines (U-2 OS, A-431, U-251MG). Each cell line was prepared with four biological replicates for total RNA and four for cytoplasmic RNA.
Publication Title
Comparison of total and cytoplasmic mRNA reveals global regulation by nuclear retention and miRNAs.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 9 Downloadable Samples
- Illumina HiSeq 2500
Description
Cell-penetrating peptides (CPP) uptake mechanism is still to be clarified to have a better understanding of their action in the mediation of oligonucleotide transfection. In this study, the effect on early events (1 h treatment) in transfection by Pepfect 14, with or without oligonucleotide cargo on gene expression, on HeLa cells, have been investigated. The RNA expression was characterized by RNA sequencing. Overall design: The quality of purified total RNA was estimated by Agilent 2200 TapeStation analysis (Agilent Technologies, Santa Clara, USA). One µg of total RNA was used as an input to prepare next-generation sequencing libraries according to the Illumina TruSeq Stranded mRNA sample preparation protocol (Illumina, San Diego, USA). Final library mixtures were quantified by Qubit 2.0 Fluorometer (Life Technologies, Grand Island, USA) and validated with Agilent 2200 TapeStation analysis. Libraries were quantified by qPCR with Kapa Library Quantification Kit (Kapa Biosystems, Woburn, USA) to optimize cluster generation and sequenced on HiSeq2500 platform (Illumina, San Diego, USA) with 2 x 50 bp paired-end reads. Over 93.9% of the bases sequenced were above the quality of Q30. Demultiplexing was done with CASAVA 1.8.2. (Illumina, San Diego, USA) Allowing one mismatch in 6 bp index read. Initial data analysis was conducted by the RNA-Seq pipeline of Estonian Genome Centre, University of Tartu. Shortly, fastQ files were trimmed (removal of adapter sequences and bases below the quality Q20) with FASTX-Toolkit version 0.013 (http://hannonlab.cshl.edu/fastx_toolkit) and then aligned to the human reference genome (hg19/GRCh37) with Bowtie version 2.1.019 in combination with TopHat version 2.0.1320. Transcript quantification (measured as FPKM) was conducted with Cuffdiff program from Cufflinks version 2.2.121 with reference annotation Homo_sapiens.GRCh37.72.gtf (http://ftp.ensembl.org/pub/release-72/gtf/homo_sapiens) Cuffdiff analysis, which summarizes expression changes for all annotated gene variations, was filtered by lowest q-values (corrected p-values for multiple testing) from output file gene_exp.diff and the top list of differentially expressed genes were analyzed through the use of QIAGEN’s Ingenuity® Pathway Analysis (IPA®, QIAGEN Redwood City, www.qiagen.com/ingenuity).
Publication Title
Role of autophagy in cell-penetrating peptide transfection model.
Sample Metadata Fields
Cell line, Treatment, Subject
View Samples