|
| Status |
Public on Apr 06, 2018 |
| Title |
Mirlet7d_Pull_Down |
| Sample type |
SRA |
| |
|
| Source name |
MLE-12
|
| Organism |
Mus musculus |
| Characteristics |
cell line: MLE-12
sample type: Pull_down
protocol: pull down experiment (Mirlet7d_Biotinylated was used for the pull down with streptavidin beads)
|
| Treatment protocol |
MLE-12 cells were transfected with 25nM of Biotinylated microRNA (Random or Mirlet7d) and harsvested after 48 h
|
| Growth protocol |
MLE-12 cells were kept at 37°c in DMEM medium containing 10 % Fetal Calf Serum, and 1% P/S/G (penicillin/Streptomycin/L-glutamine)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with TRIzol(R) (Invitrogen)
[Nuclear_RNA_seq] Libraries were prepared according to SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian (including rRNA depletion RiboGone)
[Pull_Down_Input_Mirlet7d] Libraries were treated the Kit for rRNA depletion by RiboGone™ - Mammalian and Pre-amplification step was performed with pre-amplification using SMARTer® Universal Low Input RNA Kit for Sequencing
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Ion Torrent Proton |
| |
|
| Description |
Custom LNA oligonucleotide from EXIQON
|
| Data processing |
Raw reads were visualized by FastQC to determine the quality of the sequencing. Trimming was performed using trimmomatic with the following parameters LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN
:17 CROP:60 HEADCROP:6.
High quality reads were mapped to the NONCODE V4.0 for the ncRNAs and to the GENECODE M1 version for the protein coding genes using bowtie2 with (-D 15 -R 3 -N 0 -L 20 -i S, 1, 0.56 -p8. After mapping, the sam files were converted to bam format by using samtools view (-Sb) followed by samtools sort. The sorted bam files were further converted to fastq files with SamToFastq.jar from picard-tools-1.119 and remapped to mm9 (UCSC reference genome). The tag libraries were created by using MakeTaglibrary from HOMER (-format sam). The makeUCSCfile command was used for visual
izing the mapped sequence tag density after normalizing the samples to 10 million. For the ncRNAs and for protein coding RNAs, samples were quantified by using analyzeRepeats.pl with the parameters (analyzeRepeats.pl database.gtf mm9 -count genes -s trand both and -rpkm; reads per kilobase per millions mapped). The NONCODE_V.4.gtf file was used to quantify ncRNAs and the GENECODE_M1.gtf file was used to quantify protein coding RNAs.
Mirlet7d bound ncRNAs and protein coding RNAs were identified by calculating the enrichment of the RNA-seq mapped reads (RPKM) after Mirlet7d-Pd over the input reads. KNIME 2.9.1 platform (Konstanz, Germany) was used to filter more than 2.5 times (FC>2.5) enriched ncRNAs and coding RNAs in the Mirlet7d-Pd compared to the input, by using the column filter, numeric binner and row filter nodes.
The distribution of the different ncRNA to the gene areas (Introns, Exons and 3’-UTRs) and promoter (TSS, Transcriptional Start Site, +/- 2.5 Kb) was analyzed using Bedtools v2.15 (intersect -wa -wb), crossing the two bed files, one was the bed file obtained from UCSC table browser from the mm9 promoter Refseq database track and the other file was the bed coordinate from the ncRNA, where the Mirlet7d -Pd was enriched (FC>2.5) over the input. The classification of the ncRNA was made by NONCODE name per region.
Supplementary_files_format_and_content: Bedgraph_UCSC files to load in the UCSC genome Browser
|
| |
|
| Submission date |
Dec 22, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Guillermo Barreto |
| E-mail(s) |
guillermo.barreto@univ-lorraine.fr
|
| Organization name |
CNRS Director of Research
|
| Department |
Campus Biologie-Santé. Faculté de Médecine
|
| Lab |
Laboratoire IMoPA. UMR 7365 CNRS
|
| Street address |
9, Avenue de la Forêt de Haye ‐ BP 20199
|
| City |
Nancy Cedex |
| ZIP/Postal code |
54505 |
| Country |
France |
| |
|
| Platform ID |
GPL18635 |
| Series (1) |
| GSE76248 |
MiCEE: a ncRNA-protein complex mediates epigenetic silencing and nucleolus organization |
|
| Relations |
| BioSample |
SAMN04361750 |
| SRA |
SRX1496394 |
