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Sample GSM8333036

Query DataSets for GSM8333036

Status

Public on Jun 17, 2024

Title

A2780S biol rep 1

Sample type

SRA

Source name

A2780S

Organism

Homo sapiens

Characteristics

cell line: A2780S
cell type: ovarian cancer

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated from A2780S and A2780R cells by means of NucleoSpin Total RNA kit according to the manufacturer’s protocol (Macherey-Nagel, PA, USA)
Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq X Plus

Data processing

Raw data (raw reads) of fastq format were firstly processed through fastp software. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean 1 reads were aligned to the reference genome using Hisat2 v2.0.5. We selected Hisat2 as the mapping tool for that Hisat2 can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools.
featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time.
Assembly: hg38
Supplementary files format and content: A2780S A2780R ver2.xlsx

Submission date

Jun 17, 2024

Last update date

Jun 19, 2024

Contact name

Matteo Lulli

E-mail(s)

[email protected]

Organization name

University of Florence

Street address

viale Morgagni 50