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| Status |
Public on Apr 07, 2020 |
| Title |
NC-14_2 |
| Sample type |
SRA |
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| Source name |
Human Glioma Cell Line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: U3013
treatment: NC-14
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| Treatment protocol |
4,000 cells were plated per well in a 96-well plate. The cells were treated with Ephrin-A5 DNA Nanocalipers 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. For the RNA-Seq experiment, 4,000 cells were plated in a 96-well plate format and allowed to attach for 24 hrs.
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| Extracted molecule |
total 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 NovaSeq 6000 |
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| Description |
For the DNA nanocalipers, the numbers refer to the distance (in nm) between the ligand dimers on the surface of the DNA nanocalipers. NC-0, however, refers to only one ligand dimer on the surface of the DNA nanocaliper.
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| Data processing |
Samples were sequenced on the Illumina NovaSeq 6000 platform with S1 flow cell and 2x51 setup. The Bcl to FastQ conversion was performed using bcl2fastq_v2.19.1.403 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. All genes with an RPKM value below 1 were discarded.
Differential expression analysis was tested using the DESeq2 (v. 1.24.0) R package using default settings.
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 |
GPL24676 |
| Series (2) |
| GSE138622 |
Effect of Ephrin-A5 DNA Nanocalipers stimulation of EphA2 receptor signaling in U3013 cell line |
| GSE138623 |
Ephrin-A5 modulates transcriptional responses to EphA2 activation |
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| Relations |
| BioSample |
SAMN12999569 |
| SRA |
SRX6968630 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4114470_NC-14_2.txt.gz |
195.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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