|
| Status |
Public on Mar 06, 2018 |
| Title |
Control_SMA_01 |
| Sample type |
SRA |
| |
|
| Source name |
GM03813C fibroblasts
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: GM03813C
cell type: fibroblast
disease: SMA Type I
treatment: DMSO
group: Control
concentration: --
time: --
|
| Treatment protocol |
4 total-RNA replicates of DMSO-treated GM03813C fibroblasts (control) and 4 replicates of PK4C9-treated (40 microM, 24 h)
|
| Growth protocol |
GM03813C fibroblasts were grown in MEM medium with 15% FBS, 1% penicillin/streptomycin, and 2 mM Glutamine. Cells were kept in a humidified incubator at 37 °C in 5% CO2. 4 x105 cells were seeded per well in 6-well plates (volume 2 mL) and grown to 80% confluence before treatment with PK4C9.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
GM03813C fibroblasts replicates were obtained using the RNeasy mini kit with on column DNase digestion (Qiagen). RNA concentration was quantified in a Qubit fluorometer and integrity assessed with Agilent’s 2100 bioanalyzer using pico chips. Samples with RIN values >=9.5 were sent to Beckman Coulter Genomics for RNA sequencing.
A poly-A capture protocol was applied to eliminate tRNAs and rRNAs prior to cDNA synthesis.
Eight Illumina TruSEQ RNAseq libraries were constructed and sequencing was perfomed on the Illumina HiSeq 2500 platform using paired-end 125 bp read lengths yielding a total of >400M read pairs.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Basecalling by sequencing provider
Read quality check by using FASTQC version 0.11.5
Read mapping by GSNAP version 2014-10-22 onto human genome hg19
Mapped reads per gene were determined by using human RefSeq mRNA annotations
Reads Per Kilobase of gene per Megabase of library size (RPKM) were calculated.
Reads were mapped onto RefSeq human transcripts using GSNAP version 2014-10-22 with default parameters and the option ‘sam-multiple-primaries’ in order to account for reads that map multiple times to different splice variants.
The numbers of reads mapped to exons, exon junctions, and to exons excluding junction reads was determined by applying the samtools mpileup software with default parameters for each transcript. Reads were excluded from the analysis if their start or end coordinate mapped exactly onto the splice-site.
Insert sizes of mapped read pairs determined by samtools stats software
Genome_build: hg19
Supplementary_files_format_and_content: PK4C9_counts.gct: text files in gct format containing raw read counts per gene, column 1: EntrezGene Index, column 2: Official gene symbol from EntrezGene
Supplementary_files_format_and_content: PK4C9_rpkms.gct: text files in gct format containing normalized read counts (rpkms) per gene, column 1: EntrezGene Index, column 2: Official gene symbol from EntrezGene
|
| |
|
| Submission date |
Jan 26, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Roland Schmucki |
| E-mail(s) |
roland.schmucki@roche.com
|
| Organization name |
F. Hoffmann - La Roche AG
|
| Street address |
Grenzacherstrase
|
| City |
Basel |
| ZIP/Postal code |
4058 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE94111 |
Rational targeting of RNA structure in SMN2 transcripts reverses Spinal Muscular Atrophy molecular phenotypes |
|
| Relations |
| BioSample |
SAMN06274461 |
| SRA |
SRX2520665 |
