|
| Status |
Public on Aug 31, 2018 |
| Title |
RBM7_ESC_1 [PRO-Seq] |
| Sample type |
SRA |
| |
|
| Source name |
RBM7 mESC total RNA replicate 1
|
| Organism |
Mus musculus |
| Characteristics |
cell line: ES-E14TG2a
shRNA: RBM7
cell type: Embryonic stem cells
|
| Treatment protocol |
E14 mESC were transduced with lentiviral particles. After selection with puromycin, the cells were grown in 2i media without puromycin and differentiated as follows: 1.5 million cells in 30 ml differentiation media were hung in 20 microliters drops from the lids of p15 plates with PBS in the plate for humidity. After two days, they were taken off the lid with PBS, let fall down in a falcon tube for 5 minutes. Then the liquid was removed and 26 ml of new differentiation media were added and the embryoid bodies and this media were split into two petri dishes for bacterial culture. 24 hours later, the embryoid bodies were trypsinized by incubating them for 3 minutes in 0.5% trypsin-EDTA and pipetting up and down.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Around 1 million cells were permeabilised and incubated with biotin-labeled NTPs. The RNA was then hydrolyzed with NaOH and biotin containing fragments were enriched by affinity purification using streptavidin-coated magnetic beads.
A 3′ sequencing adaptor was ligated to the hydroxyl (OH) group at the 3′ end of nascent RNA, the 5'cap was removed, then a 5'OH was generated by base hydrolysis and converted to 5′ phosphate by treatment with T4 polynucleotide kinase. Then, a 5′ sequencing adaptor was ligated to the nascent RNA. RNA was purified to enrich for fragments with adaptor at both ends and reverse transcribed.
PROseq. Libraries were sequenced on an Illumina NextSeq 500 (75 bp – high throughput kit).
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
ES cells grown in 2i medium and transduced with a pLKO lentiviral vector carrying a shRNA against RBM7 (NM_144948.2-1021s1c1).
|
| Data processing |
Library strategy: PRO-Seq
Adapter sequences were trimmed with FASTX-Toolkit and filtered for a minimum of 15 bases. All reads were then trimmed to a maximum of 36 bases and then reverse complimented.
Reads were first mapped with Bowtie to a copy of the mouse rDNA repeat (GenBank: BK000964.1) with the -K1 option, and unaligned reads were then mapped to the mouse genome (mm10) filtering for unique matches.
Uniquely mapped reads were used for downstream analysis.
Only the final 3’-base representing position of the polymerase was reported to output files used in all analyses.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files were generated using Bedtools and bedGraphtoBigWig. The score represents strand specific per base genome coverage.
|
| |
|
| Submission date |
Aug 02, 2018 |
| Last update date |
Sep 02, 2018 |
| Contact name |
Evdoxia Karadoulama |
| E-mail(s) |
evka@mbg.au.dk
|
| Organization name |
Aarhus University
|
| Department |
Molecular Biology and Genetics
|
| Lab |
Torben Heick Jensen
|
| Street address |
C.F. Møllers Alle 3
|
| City |
Aarhus |
| ZIP/Postal code |
8000 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE115727 |
The RNA exosome contributes to gene expression regulation during stem cell differentiation |
| GSE118052 |
The RNA exosome contributes to gene expression regulation during stem cell differentiation [PRO-Seq 2] |
|
| Relations |
| BioSample |
SAMN09761011 |
| SRA |
SRX4502325 |
