|
| Status |
Public on Jul 15, 2024 |
| Title |
RNAseq-DOX-EtOH-N2 |
| Sample type |
SRA |
| |
|
| Source name |
SH-EP NMYC-ER
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SH-EP NMYC-ER
cell type: Neuroblastoma
dox-treatment (shrna-tf3c5): induced (shRNA expression)
4-oht treatment_(nmyc_expresion): uninduced (no NMYC expression)
treatment with_inhibitor: not applicable
biological replicate: N2
|
| Treatment protocol |
The indcution of NMYC in the SH-EP-NMYC-ER cells was achieved by adding 4-OHT (200 nM) for 4 hours. The expression of an shRNA targeting TF3C5 in the SHEP-NMYCER cells was induced with 1 µg/ml doxycycline for 48 hours.
|
| Growth protocol |
Human neuroblastoma derived SH-EP cells expressing an exogenous and 4-OHT inducible NMYC allele (SH-EP-NMYC-ER), were grown in RPMI-1640 medium supplemented with with 10% horse serum and 1% penicillin/ streptomycin. Cells were cultured at 37C at 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA sequencing, cells were harvested and total RNA was extracted with RNeasy MiniKit (Qiagen) according to manufacturer's instructions. On-column Dnase I digestion was performed followed by mRNA isolation with the NEBNext Poly(A) mRNA Magnetic Isolation Kit (NEB).
Library preparation was done with the NEBNext Ultra II Directional RNA Library Prep for Illumina (NEB) following the manufacturer's manual. Libraries were size-selected using SPRIselect beads (Backman-Coulter) after amplification with 9 PCR cycles using the NEBNext Multiplex Oligos for Illumina Kit (NEB). Library quality and fragment size distribution was analyzed on a Fragment Analyzer (Advanced Analytical). Sequencing was performed in paired-end mode on a NextSeq2000 platform (Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
| |
|
| Description |
RNA-seq
|
| Data processing |
Basecalls were performed using bcl2fastq v1.1.0 (Illumina) and the quality of the generated FASTQ files was analysed with FastQC.
For mRNA sequencing, reads were aligned to the human genome (hg19/GRCh37) using STAR aligner v2.7.9a with default parameters and gene quantification (--quantMode GeneCounts). The output table with teh gene counts was futher analyzed in R v4.1.1 and processed with DESeq2 package according to the manual. Genes with at least 15 counts in at least 3 samples were kept in the counttable. All samples were then normalized by size factors.
Following read-normalization, the fold average for 3 independent biological replicates (N1, N2, N3) was calculated on treatment to control. Where applicable, the calculated fold-changes were averaged in 100 genes per bin for a total of 140 bins of 14085 genes.
Assembly: hg19
Supplementary files format and content: Tab-delimited text file that includes the raw-counts for all 12 individual samples in a singel table. This count-table was used to carry out all further data-processing steps as outlined above.
|
| |
|
| Submission date |
Jan 17, 2023 |
| Last update date |
Jul 15, 2024 |
| Contact name |
Martin Eilers |
| Organization name |
University of Wuerzburg
|
| Department |
Chair for Biochemistry and Molecular Biology
|
| Lab |
Martin Eilers
|
| Street address |
Am Hubland
|
| City |
Wuerzburg |
| ZIP/Postal code |
97074 |
| Country |
Germany |
| |
|
| Platform ID |
GPL30173 |
| Series (2) |
| GSE223057 |
TFIIIC and MYCN link the three-dimensional chromatin structure of promoters to transcription termination of stalled RNA polymerase (RNA-Seq) |
| GSE223058 |
TFIIIC and MYCN link the three-dimensional chromatin structure of promoters to transcription termination of stalled RNA polymerase |
|
| Relations |
| BioSample |
SAMN32769630 |
| SRA |
SRX19051683 |
