|
| Status |
Public on Jul 20, 2015 |
| Title |
FTO3C3_Input |
| Sample type |
SRA |
| |
|
| Source name |
HEK293 derivative
|
| Organism |
Homo sapiens |
| Characteristics |
rna type: small RNA
treatment of cells: no treatment
cell line: HEK293 derivative
|
| Treatment protocol |
Cells were transfected following the standard protocol at 10 nM concentration of siRNAs using lipofectamine 2000 as a transfection reagent. Briefly, cells were plated in 6-well plate one day before transfection. On day of transfection, the medium was changed, and 2 h later the formed complexes of siRNAs and lipofectamine were added to cells in a drop-like manner. RNA and protein were extracted after 48 h of transfection from both, FTO siRNA and scrambled siRNA transfected cells.
|
| Growth protocol |
Cells were maintained in DMEM medium supplemented with 10% FCS and 1% PenStrep in a humidified incubator at 37°C supplied with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The large and small RNA fractions from FTO knockdown was prepared using RNeasy Plus Universal mini kit (Qiagen, Hilden, Germany) following the instructions of the manufacturer. The small RNA fraction was eluted with the RNeasy MinElute Cleanup Kit (Qiagen, Hilden, Germany).
Illumina TruSeq Small RNA Sample Preparation Guide was used for cDNA synthesis and library preparation. The protocol was strictly followed. Briefly, 3’ and 5’ adapters were ligated to the samples, followed by reverse transcription and amplification with primers containing the unique indices for different samples. Amplified cDNA constructs were purified using Ampure XP beads. DNA was loaded into two lanes at 5 pM and 10 pM concentrations and subjected to Illumina sequencing. Demultiplexing was done using Illumina CASAVA 1.8.2.
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Illumina TruSeq adapters were removed with cutadapt; reads with remaining length 16-28 were kept as potential miRNAs, full length reads as potential other RNAs
Potential miRNAs were mapped against miRbase v20 (July 2013) using mamaslemonpy (in-house software, available on Python Package Index PyPI), counts were tabulated
Whole workflow is implemented in Snakemake and is available as a Snakefile
Genome_build: mirBase v20
Supplementary_files_format_and_content: Two excel files showing the read counts of miRNAs in FTO ko and RNA immunoprecipitation experiments
|
| |
|
| Submission date |
Jul 25, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Ludger Klein-Hitpass |
| E-mail(s) |
ludger.klein-hitpass@uni-essen.de
|
| Phone |
+49 201 723 85552
|
| Organization name |
Institut fuer Zellbiologie
|
| Department |
Universitaetsklinikum
|
| Lab |
BioChip Lab
|
| Street address |
Virchowstr. 173
|
| City |
Essen |
| ZIP/Postal code |
D-45122 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE59767 |
N6-adenosine methylation in miRNAs |
|
| Relations |
| BioSample |
SAMN02940959 |
| SRA |
SRX661491 |
