|
| Status |
Public on Jan 04, 2016 |
| Title |
rep1_siCtrl_for_MYC |
| Sample type |
SRA |
| |
|
| Source name |
IMEC cell line
|
| Organism |
Homo sapiens |
| Characteristics |
sirna: siCtrl
replicates: 1
|
| Treatment protocol |
Cells were infected with the indicated doxycycline-inducible constructs and treated with Dox or EtOH as solvent control or transfected with the indicated siRNAs.
|
| Growth protocol |
IMEC cell lines were grown in DMEM/F-12 with appropriate supplements.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Mini Kit (Qiagen) including on-column DNase digestion. Poly-A RNA was isolated from total RNA using the NEBNext Poly(A)mRNA Magnetic Isolation Module (E7490). For 4-SU seq, nascent RNA transcripts were labelled for 10 min using 1 mM 4-thiouridine (4sU, Sigma T4509). After RNA isolation the 4SU-labelled RNAs were biotinylated and enriched using MyOne Streptavidin beads (Life Technologies). Eluted RNAs were subsequently used for library preparation using NEBNext RNA-Sequencing kits.
Libraries for the RNA-seq samples were constructed using the NEBNext Library Prep Kit (6100) following the instruction manual. Briefly, poly-A RNA was fragmented to generate 200 nucleotides long fragments. First and second strand cDNA synthesis was performed and the resulting cDNA was end-repaired, ligated to NEBNext adaptors. The cDNA was size selected using agarose gels and amplified by PCR. For the resulting RNA-seq libraries, amplicon sizes and quantities were determined using the Biorad Experion system. Libraries were sequenced on an Illumina NextSeq 500 following the manufacture’s instructions.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
polyA RNA
norm_count_table_for_siMYC_siCDC73.csv
|
| Data processing |
Basecalling and Demultiplexing was performed using BaseSpace from Illumina.
Reads were aligned to the human genome (hg19) with BOWTIE2 v2.1.0 using standard-options
Bedgraph files were generated using Bedtools
Differential expression and statistical inference was done using edgeR.
Genome_build: hg19
Supplementary_files_format_and_content: The csv-files contain a table showing the edgeR normalized counts for each transcript. The bedgraph files were generated using Bedtools for each sample.
|
| |
|
| Submission date |
Nov 20, 2015 |
| Last update date |
May 15, 2019 |
| 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 |
GPL18573 |
| Series (2) |
| GSE70000 |
Ubiquitin-dependent turnover of MYC promotes loading of the PAF complex on RNA Polymerase II to drive transcriptional elongation (RNA-seq) |
| GSE70009 |
Ubiquitin-dependent turnover of MYC promotes loading of the PAF complex on RNA Polymerase II to drive transcriptional elongation |
|
| Relations |
| BioSample |
SAMN04285080 |
| SRA |
SRX1441820 |
