|
| Status |
Public on Jan 28, 2013 |
| Title |
KO |
| Sample type |
SRA |
| |
|
| Source name |
embryonic heart ventricles
|
| Organism |
Mus musculus |
| Characteristics |
genetic background: C57BL/6 x 129SV
genotype: Fendrr KO
developmental stage: E12.5
tissue: heart ventricle
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from 4 pooled ventricles of each WT and KO samples using the RNAeasy Mini Kit (Qiagen). Ribosomal RNA from 500ng of total RNA was depleted using the Ribo-Zero Magnetic Kit (Epicentre) according to manufacturer’s instructions with minor modifications to accommodate for input amounts lower than 1µg. In brief, we used 90µl of beads per reaction, washed them with an equal amount of H2O and resuspended them in 35µl of resuspension solution with the addition of 0.5µl of RNase inhibitor. We added 2µl of rRNA removal solution and 2µl of reaction buffer to 16µl of the total RNA (corresponding to 500ng). The final eluate was cleaned-up using RNA MinElute columns (Qiagen) and eluted in 10µl H2O.
The complete eluate was used to prepare sequencing libraries using the Script-Seq v2 RNA-Seq Library Preparation Kit (Epicentre) according to manufacturer’s instructions. The cDNA was purified using DNA MinElute columns (Qiagen), 15 cycles of PCR were used to amplify the library using index primers and the final library was purified using AMPure XP beads (Beackman Coulter). The libraries were sequenced on two independent runs using the MiSeq v2 (Illumina) running at 1x100bp cycles.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
Embryos obtained from tatraploid complementation of embryonic stem cells in which Fendrr was knocked-out on both alleles via homologous recombination. The first run (WT) was originally started as a 2x100bp run, but the run aborted during the ~20th cycle of the second mate. The base-calling of the first 100bp was then performed manually by Illumina (because the automated MiSeq pipeline was not doing it after aborting the run), at which point it is possible that they applied a 100bp filter. For the second sample (KO) to be comparable, only a 1x100bp run was performed which finished without problems and the normal MiSeq pipeline was performing the base-calling.
|
| Data processing |
The raw reads were aligned against the mouse genome (mm9) using Tophat (version 2.0.6) and Bowtie2 (version 2.0.2), supplying the UCSC refseq transcriptome annotation and using the following parameters: --coverage-search --library-type fr-secondstrand --b2-very-sensitive --no-novel-juncs --no-novel-indels
We used Cufflinks (version 2.0.2) to calculate transcript coverage and abundance (FPKM) across the UCSC mm9 refseq transcripts and combined all isoforms into one metagene.
Genome_build: mm9
Supplementary_files_format_and_content: Tab-seperated file containing gene symbols and the corresponding WT and KO FPKM values.
|
| |
|
| Submission date |
Dec 20, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Frederic Koch |
| Organization name |
Max Planck Institute for Molecular Genetics
|
| Department |
Developmental Genetics
|
| Street address |
Ihnestraße 63-73
|
| City |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16417 |
| Series (1) |
| GSE43078 |
The tissue-specific lncRNA Fendrr is an essential regulator of heart and body wall development in the mouse |
|
| Relations |
| SRA |
SRX212199 |
| BioSample |
SAMN01832337 |
