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| Status |
Public on Jan 11, 2022 |
| Title |
3_1483co_IGF1 |
| Sample type |
SRA |
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| Source name |
glioma cells co-cultured with OB neurons after IGF-1 stimulation
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| Organism |
Mus musculus |
| Characteristics |
cell type: glioma
tissue: brain
genotype: p53-/-;NF1-/-;tdTomato+/-
treatment: IGF-1 stimulation
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| Treatment protocol |
Experiment group was incubated with 20 ng/mL IGF-1 overnight.
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| Growth protocol |
OBs were dissected from P2~3 mouse pups and digested in 10 mL papain solution (1x EBSS (Sigma, E7510), 0.5 mM EDTA, 10 mM HEPES, 26 mM NaHCO3, 22.5 mM D(+)-glucose, 0.16 mg/mL L-cycteine, 20 units/mL papain (Worthington Biochem, LS003126), 250 μg/mL DNase I (Sigma, DN-25) in dd-water) for 30min at 37°C. Papain solution was discarded and the tissue was triturated with 2 mL Diluted Trypsin Inhibitor Buffer (1x EBSS (Sigma, E7510), 10 mM HEPES, 26 mM NaHCO3, 1x ovomucoid (Worthington Biochem, LS003086), 250 μg/mL DNase I (Sigma, DN-25), 1 mg/mL BSA (equitech-bio, BAH62) in dd-water) by 1 mL pipette and was allowed to settle for 2 min. 1 mL upper cell suspension was carefully collected and additional 1 mL Diluted Trypsin Inhibitor Buffer was add to the tissue. The process was repeated until all Diluted Trypsin Inhibitor Buffer was consumed. Cell suspension was layered over the Standard Trypsin Inhibitor Buffer (1x EBSS (Sigma, E7510), 10 mM HEPES, 26 mM NaHCO3, 1x ovomucoid (Worthington Biochem, LS003086), 10 mg/mL BSA (equitech-bio, BAH62) in dd-water) and centrifuged at 1,200 rpm for 10 min. Supernatant was discarded and cells were resuspended with plating medium (25 mM D(+)-glucose, 2.5 mM NaHCO3, 0.1 mg/mL transferrin (Sigma, T-1147), 10% FBS (serapro, s601s), 2 mM L-glutamine, 0.25 mg/mL insulin (Sigma, I-6634), 1x pen/strep (Hydone, SV30010), 50 μg/mL gentamycin (Sigma, 46305) in neurobasal medium (Gibco, #12349015)) for cell culture. 24 h after initial plating, 80% medium was removed and replaced with 0ARAC medium (25 mM D(+)-glucose, 2.5 mM NaHCO3, 0.1 mg/mL transferrin (Sigma, T-1147), 5% FBS (serapro, s601s), 0.5 mM L-glutamine, 1x pen/strep (Hydone, SV30010), 50 μg/mL gentamycin (Sigma, 46305), 1x B27 (Gibco, 17504044) in neurobasal medium (Gibco, #12349015)). 48-72 h after initial plating, 50% medium was removed and replaced with 4ARAC medium (25 mM D(+)-glucose, 2.5 mM NaHCO3, 0.1 mg/mL transferrin (Sigma, T-1147), 5% FBS (serapro, s601s), 0.5 mM L-glutamine, 1x pen/strep (Hydone, SV30010), 50 μg/mL gentamycin (Sigma, 46305), 1x B27 (Gibco, 17504044), 4 μM AraC (MKbio, MS0237) in neurobasal medium (Gibco, #12349015)). Glioma cells were plating on OB neurons for co-culture on DIV7 with experiment medium (25 mM D(+)-glucose, 2.5 mM NaHCO3, 0.5 mM L-glutamine, 1x pen/strep (Hydone, SV30010), 50 μg/mL gentamycin (Sigma, 46305), 1x B27 minus insulin (Gibco, A1895601), in neurobasal medium (Gibco, #12349015)).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Glioma cells were seperated from neurons by FACS through tdTomato tag In the tuomor. RNA was harvested using Trizol reagent.
RNA libraries were prepared for sequencing using standard Illumina protocols
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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 |
raw data was filtered by removing adapter with Cutadapt (Version 3.4) and quality control (not lower than 25).
Clean reads were aligned to the reference genome Mus_musculus.GRCm39.dna.primary_assembly.fa (39.103) from Ensembl by STAR (Version 2.7.9a).
Read counts were calculated by featureCounts (Version 2.0.1). The feature was selected as Exon, and only reads with mapping quality score greater than 25 were retained.
The batch effect was removed by sva (Version 3.63.0) for samples sequenced at different times.
Differential gene expression analysis was performed by DESeq2 (Version 1.28.1). The screening threshold was fold change > 1.5 and P-adj < 0.05.
Genome_build: GRCm39
Supplementary_files_format_and_content: tab-delimited text files include count values for each Sample
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| Submission date |
Dec 01, 2021 |
| Last update date |
Jan 11, 2022 |
| Contact name |
Chong Liu |
| E-mail(s) |
0015027@zju.edu.cn
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| Organization name |
Medical College of Zhejiang University
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| Street address |
NO.866 of Yuhang Tong Road
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| City |
Zhejiang |
| ZIP/Postal code |
310058 |
| Country |
China |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE189940 |
Next Generation Sequencing Analysis of Mice Glioma Cells co-cultured with OB Neurons |
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| Relations |
| BioSample |
SAMN23551206 |
| SRA |
SRX13273277 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5710232_3_1483co_IGF1.txt.gz |
160.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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