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Sample GSM4815230

Query DataSets for GSM4815230

Status

Public on Dec 15, 2021

Title

siNPM1 Hypoxia rep2

Sample type

SRA

Source name

HeLa cell line

Organism

Homo sapiens

Characteristics

cell line: HeLa
knockdown: NPM1 siRNA
treatment: 1% Oxygen

Treatment protocol

HeLa cells treated with control (Nt) or NPM1 siRNA (siNPM1) for 24h (Viromer reagent transfection)

Growth protocol

HeLa cells treated with control (Nt) or NPM1 siRNA (siNPM1) for 24h and incubated at 21% or 1% O2 for 24h

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted with nucleozol reagent
Total RNA samples were prepared and their concentration was measured in nanodrop (ND1000 Spectrophotometer – PEQLAB). The concentration required for library preparation varies from 5 to 500ng per 5μl. Samples with a concentration of more than 500ng/μl, were diluted accordingly to a mean concentration of approximately 100-150ng/μl. The RNA quality of each sample was measured in bioanalyzer (Agilent Technologies) using the Agilent RNA 6000 Nano Kit reagents and protocol. (RNA samples with RNA Integrity Number (RIN) >7 are preferred, although RNAs of lower quality can also be used, and further processed for library preparation and sequencing). For library preparation, the 3’ mRNA-Seq Library Prep Kit Protocol and reagents, for Ion Torrent (QuantSeq-LEXOGEN™ Vienna, Austria) was used according to manufacturer’s instruction. Briefly, library generation is initiated by oligodT priming which contains the Ion Torrent compatible linker sequences. Approximately 300ng per 5μl of RNA from each sample was used to perform the first strand synthesis. Any remaining RNA is removed and 2nd strand synthesis is initiated by a random primer, containing Ion Torrent compatible linker sequences at its 5’ end, and a sequence polymerase. In line barcodes are introduced at this point. 2nd strand synthesis is followed by a magnetic bead-based purification step and the resulted purified library is amplified for 13 to 18 cycles and re-purified. Each library’s quality and quantity was assessed in bioanalyzer using the DNA High Sensitivity Kit reagents and protocol (Agilent Technologies). The quantified libraries were pooled together in 12plex at a final concentration of 7pM. The libraries pool was processed on the Ion Proton One Touch system where the libraries were templated and enriched with the Ion PI™ Hi-Q™ OT2 200 Kit (ThermoFisher Scientific) and sequenced, with the Ion PI™ Hi-Q™ Sequencing 200 Kit on Ion Proton PI™ V2 chips (ThermoFisher Scientific) according to commercially available protocols. 3’RNA-sequencing is performed on an Ion Proton™ System (Rothberg et al, 2011), according to the manufacturer's instructions. Initial analysis took place in Ion Torrent server where the strand specific sequencing reads mapped to their corresponding strand on the genome.
3’RNA-sequencing

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Ion Torrent Proton

Data processing

Base calling was performed using software provided with the Ion Torrent Proton sequencer.
Sequenced reads were trimmed for adaptor sequence using cutadapt software and mapped to hg19 whole genome using tophat 2.0.9 with default settings and using additional transcript annotation data for the mm9 genome from Illumina iGenomes (http://cufflinks.cbcb.umd.edu/igenomes.html). Tags overlapping the Ensembl GRCh37 human exons were counted and filtered for artifacts as follows: if an annotated gene had up to five exons, tag presence was required in at least two exons. If an annotated gene had E >5 exons, tag presence was required in at least E/5 exons. The final gene counts were calculated as the sums of their exon tags. The counts table was normalized and analyzed for differential expression using DESeq. The final list of differentially expressed genes was derived by the genes demonstrating a binomial test p-value less than 0.05.
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited files containing DESeq normalized counts for each sample and Ensembl gene.

Submission date

Oct 01, 2020

Last update date

Dec 15, 2021

Contact name

Antonis Giakountis

Organization name

University of Thessaly

Department

Biochemistry and Biotechnology

Lab

Laboratory of Molecular Biology and Genomics