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Status |
Public on Jan 19, 2024 |
Title |
A549 cells, CST1, rep2 |
Sample type |
SRA |
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Source name |
A549
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Organism |
Homo sapiens |
Characteristics |
cell line: A549 cell type: adenocarcinomic human alveolar basal epithelial cells genotype: WT treatment: CST1
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Treatment protocol |
transfected with CST1 plasmid with the Lipofectamine reagent, the RNA was extracted 48 hours after transfection
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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.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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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
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Submission date |
Apr 14, 2023 |
Last update date |
Jan 19, 2024 |
Contact name |
Angela Hoyer |
Organization name |
Karolinska Institutet
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Street address |
Visionsgatan 4
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City |
Solna |
ZIP/Postal code |
171 76 |
Country |
Sweden |
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Platform ID |
GPL24676 |
Series (1) |
GSE229741 |
The functional role of CST1 and CCL26 in asthma development |
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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 Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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