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Sample GSM8564872

Query DataSets for GSM8564872

Status

Public on Oct 11, 2024

Title

HK2 cells, AdSirt4+TGF 1

Sample type

SRA

Source name

Kidney

Organism

Homo sapiens

Characteristics

tissue: Kidney
cell line: HK5 cells
cell type: Tubular epithelial cells
genotype: WT
treatment: TGF-beta1 stimulation

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated using the Trizol Reagent (Invitrogen Life Technologies), after the total RNA was qualified and quantified as follows: (1) RNA purity and concentration were then examined using NanoDrop spectrophotometer (Thermo Scientific) and Qubit 4.0; (2) RNA integrity and quantity were measured using the Agilent 2100/4200 system.
Three micrograms of RNA were used as input material for the RNA sample preparations. Sequencing libraries were generated according to the following steps. Firstly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in fragmentation buffer. First strand strand was synthesized by random hexamers, and the second cDNA strand was synthesized by adding buffer, dNTPs, RNase H and DNA polymerase I. Double stranded cDNA was repaired and A was added to the 3 'end. Hieff NGS® DNA Selection Beads were used for purification and fragment Selection. After purification and fragment selection, the products were amplified and enriched by PCR. Qubit was used for quantitative .
The target region library of the duplex was denatured, cycled, and digested to yield a single-stranded circular DNA. Single-stranded circular DNA is amplified by a rolling circle amplification (RCA), known as DNA nano balls (DNB). After library construction, Qubit was used for quantitative.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

DNBSEQ-T7

Data processing

Samples are sequenced on the platform to get image files, which are transformed by the software of the sequencing platform, and the original data in FASTQ format (Raw Data) is generated. Sequencing data contains a number of connectors, low-quality Reads, so we use fastp (v0.21.0) software to filter the sequencing data to get high quality sequence （ Clean Data ） for further analysis .
The reference genome and gene annotation files were downloaded from genome website. The filtered reads were mapping to the reference genome using HISAT2 v2.1.0.
We used StringTie(v2.1.5) statistics to compare the Read Count values on each gene as the original expression of the gene, and then used FPKM to standardize the expression. Then difference expression of genes was analyzed by DESeq2 (v1.30.1) with screened conditions as follows: expression difference multiple |log2FoldChange| > 1, significant padj≤0.05. At the same time, We used R language Pheatmap(1.0.8) software package to perform bi-directional clustering analysis of all different genes of samples. We geted heatmap according to the expression level of the same gene in different samples and the expression patterns of different genes in the same sample with Euclidean method to calculate the distance and Complete Linkage method to cluster.
Assembly: hg38
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample

Submission date

Oct 10, 2024

Last update date

Oct 11, 2024

Contact name

Shu Yang

E-mail(s)

yang.shu@szhospital.com

Organization name

shenzhen people's hospital

Street address

1017 Dongmen North Road, Luohu District, Shenzhen