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| Status |
Public on Apr 07, 2020 |
| Title |
MDA_treatment_2 |
| Sample type |
SRA |
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| Source name |
Human Breast Adenocarcinoma Cell Line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MDA-MB-231
treatment: IgG-clustered Ephrin-A5
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| Treatment protocol |
For both cell lines, 4,000 cells were plated per well in a 96-well plate. The cells were treated with either IgG or IgG-clustered Ephrin-A5 (ICE) in a total volume of 50 µl for 30 min at 37℃, 5% CO2. This was performed in duplicates which were pooled together after cell lysis.
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| Growth protocol |
For the U3013 cell line, a T25 flask was coated with 10 µg/ml of poly-L-ornithine (Sigma-Aldrich) and allowed to incubate for 3 h at room temperature.After 3 h, the flask was washed with twice with 1XDPBS (Gibco). The plate was then coated with 10 µg/ml laminin (Sigma-Aldrich) and allowed to incubate for 30 min at 37°C, 5% CO2. After incubation, most of the laminin was removed. The U3013 cell line was cultured in coated T25 flasks in GBM media (For 50 ml: 25 ml Neurobasal media (Thermofisher Scientific), 25 ml DMEM/F-12, GlutaMAX suppl. (ThermoFisher Scientific), 10 ng/ml EGF (R&D Systems), 10 ng/ml bFGF (ThermoFisher Scientific), 1 ml B27 (ThermoFisher Scientific) and 0.5 ml N2 Supplement (ThermoFisher Scientific) supplemented with 1% penicillin/streptomycin. 1x10E6 U3013 cells were then added to the T25 flask. The MDA-MB-231 Cell Line (ATCC) was cultured in Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10 % fetal bovine serum and 1% penicillin/streptomycin. For the RNA-Seq, 4,000 cells were plated in a 96-well plate format and allowed to attach for 24 hrs, MDA-MB-231 were subjected to serum starvation and U3013 were seeded onto the coated wells, as detailed above.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
The cells were lysed according to the SMART-Seq2 protocol
The Smart-Seq2 protocol was used for the library construction with minor changes. After stimulation and lysis, rather then using 2 µl of the cell lysate, 5 µl was used and subsequent steps, ie. reverse transcription and PCR preamplification, were performed in twice the volume, 20 and 50 µl instead of 10 and 25 µl, to prevent any inhibitory effects from occurring. For the tagmentation step, 200 pg of cDNA was used for input. The final libraries for both experiments were prepared utilizing Illumina Paired-end sequencing using the Nextera XT library prep kit (Illumina). The library was sequenced on the HiSeq2500 platform with a 2x126 setup using 'HiSeq SBS Kit v4' chemistry
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Clustering was done by 'cBot' and samples were sequenced on HiSeq2500 (HiSeq Control Software 2.2.58/RTA 1.18.64) with a 2x126 setup using 'HiSeq SBS Kit v4' chemistry. The Bcl to FastQ conversion was performed using bcl2fastq_2.19 from the CASAVA software suite. The quality scale used is Sanger / phred33 / Illumina 1.8+.
Sequenced reads were trimmed for adaptor sequence and low quality bases using Trimmomatic (v. 0.35) for paired-end reads using the following parameters: -phred33 ILLUMINACLIP:'./adapters/NexteraPE.fa':2:30:10 TRAILING:15 SLIDINGWINDOW:4:18 MINLEN:36.
Trimmed RNA-Seq reads were mapped to the GRCg38 genome using the STAR package (v. 2.2.1) using default settings
Count tables were generated for each mapped sample using htseq-count (v. 0.5.4p3). This was performed using default settings except for the following: --stranded= No, and all reads below an alignment quality of 10 were skipped.
Differential expression analysis was tested using the DESeq2 (v. 1.24.0) R package using default settings. For the analysis, both paired and single reads (whose pair was not of sufficient quality) were used.
Genome_build: GRCh38
Supplementary_files_format_and_content: tab-delimited text files containing the count tables per sample generated by the htseq-count package
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| Submission date |
Oct 09, 2019 |
| Last update date |
Apr 07, 2020 |
| Contact name |
Toon Verheyen |
| E-mail(s) |
Toon.verheyen@ki.se
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| Organization name |
Karolinska Institutet
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| Department |
Medical Biochemistry and Biophysics
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| Lab |
Teixeira Lab
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| Street address |
Tomtebodavägen 16
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| City |
Solna |
| ZIP/Postal code |
17165 |
| Country |
Sweden |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE138620 |
Effect of IgG-clustered Ephrin-A5 stimulation on EphA2 signaling in MDA-MB-231 and U3013 cell lines |
| GSE138623 |
Ephrin-A5 modulates transcriptional responses to EphA2 activation |
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| Relations |
| BioSample |
SAMN12999552 |
| SRA |
SRX6968612 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4114447_MDA_treatment_2.txt.gz |
186.0 Kb |
(ftp)(http) |
TXT |
| GSM4114447_MDA_treatment_R1_single_2.txt.gz |
177.9 Kb |
(ftp)(http) |
TXT |
| GSM4114447_MDA_treatment_R2_single_2.txt.gz |
168.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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