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Sample GSM7174480 Query DataSets for GSM7174480
Status Public on Jan 19, 2024
Title A549 cells, CST1, rep2
Sample type SRA
 
Source name A549
Organism Homo sapiens
Characteristics cell line: A549
cell type: adenocarcinomic human alveolar basal epithelial cells
genotype: WT
treatment: CST1
Treatment protocol transfected with CST1 plasmid with the Lipofectamine reagent, the RNA was extracted 48 hours after transfection
Extracted molecule total RNA
Extraction protocol Expression plasmids containing the human CST1 and CCL26 as well as the empty vector pCMV6-entry as control were obtained from OriGene Technologies Inc. (USA, Table 1). A549 cells were transfected with 1 µg plasmid DNA containing CST1 (CST1 overexpression) or CCL26 (CCL26 overexpression) separately in 24-well plates using Lipofectamine® 3000 (Thermofisher Scientific, USA), according to the manufacturer`s instruction. The cells were harvested for subsequent experiments 48 hours following transfection. All experiments were performed in biological quadruplicates. Total RNA was extracted with the Qiagen RNeasy Micro kit (Hilden, Germany) 48 hours after transfection and the concentrations were measured using QubitTM Flex Fluorometer (Thermofisher Scientific, USA). RIN values were obtained from Qsep100 Bio-Fragment Analyzers (Bioptic, Inc., Taiwan) and ranged between 8.9-10. Overexpression was confirmed using rt-qPCR (supplementary Figure 1). cDNA was synthesized using SuperScript III First-Strand Synthesis SuperMix (Thermofisher Scientific, USA), according to the manufacturer`s protocol. For rt-qPCR, 1 µl of cDNA was used in a 20 µl reaction volume, TaqMan gene expression Master Mix and pre-developed TaqMan gene expression assays (Thermofisher Scientific, USA). All samples were run in triplicates according to the manufacturer`s instructions. Each reaction was normalized to the GAPDH and PPIA content separately and the ΔΔCT method was used to determine the fold induction over cells transfected with the empty vector (control samples).
In total, 12 samples were included (4 samples CST1, 4 samples CCL26, 4 samples pCMV6-Entry). The libraries were prepared with the Illumina® TruSeq Stranded mRNA library preparation (Bravo) kit with 330 ng of each sample. The average library length for CST1 was equal to 362 bp, for CCL26 equal to 400 bp and for pCMV6-entry equal to 395 bp. Sequencing was performed on an IlluminaⓇ NovaSeq 6000 platform using the NovaSeq 6000 S4 Reagent Kit (35 cycles) (Illumina, San Diego, CA, USA). Sequencing was performed at the National Genomics Infrastructure in Stockholm, Sweden.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Data processing During RNA sequencing (RNAseq) data preprocessing, the quality of the raw RNAseq reads was evaluated using FastQC (v0.11.1, Babraham Bioinformatics, UK) and then trimmed accordingly by Trim Galore (v0.6.1, Babraham Bioinformatics, UK) to achieve good quality reads with a Phred score of Q30. Cleaned reads were mapped to the human reference transcriptome (GRCh38.p13) downloaded from Ensembl (release 105, downloaded March 2022) to estimate the gene expression by Kallisto(24). Using R and Bioconductor packages, TPM (transcripts per million) values for each protein-coding transcript were extracted, collapsed at the gene level (with gene symbol), and analyzed for differential expression using edgeR (version 3.32.1). A generalized linear model design = ~ Cell-type (Treated (overexpressed) or non-treated (control)) was used to calculate differential gene expression between treated and non-treated cells. Log2 transformed fold change, p-value, FDR were calculated for each gene. We considered |log2FC| ≥ 2 and FDR < 0.05 as the cutoff values to identify differentially expressed genes (DEG).
Assembly: human reference transcriptome (GRCh38.p13)
Supplementary files format and content: TSV-fil (.tsv) include TPM values
 
Submission date Apr 14, 2023
Last update date Jan 19, 2024
Contact name Angela Hoyer
Organization name Karolinska Institutet
Street address Visionsgatan 4
City Solna
ZIP/Postal code 171 76
Country Sweden
 
Platform ID GPL24676
Series (1)
GSE229741 The functional role of CST1 and CCL26 in asthma development
Relations
SRA SRX19972496
BioSample SAMN34191133

Supplementary file Size Download File type/resource
GSM7174480_P23757_102.tsv.gz 2.6 Mb (ftp)(http) TSV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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