|
| Status |
Public on Apr 06, 2018 |
| Title |
Ctrl_PRO-seq_MLE_12 |
| Sample type |
SRA |
| |
|
| Source name |
MLE-12
|
| Organism |
Mus musculus |
| Characteristics |
cells: MLE-12
sample type: control
|
| Treatment protocol |
MLE-12 cells were transfected with 2.5ug of empty vector of Mirlet7 sponge construct and harsvested after 48 h.
|
| Growth protocol |
Mouse lung epithelial cell MLE-12 (ATCC CRL-2110) was kept at 37°c in DMEM: F12 medium containing 5 % Fetal Calf Serum, and 1% P/S (penicillin/Streptomycin). Schneider’s Drosophila Line 2 (S2 cell line, ATCC CRL-1963) was cultured in complete Schneider’ Drosophila medium (10% heat inactivated FCS, 1% Penn-strep) at 26 deg C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
MLE-12 cells were harvested and spike-in control (Drosophila S2 cells) were added just before cell permeabilization. Then cells were permeabilized. Precision nuclear run-on was performed. Biotinylated nascent RNAs were enriched and purified using Trizol (Invitrogen #15596026).
Library preparation of nuclear RNA was performed using Lib-Prep: SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian (Clontech) following the manufacturer’s instructions.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Visualization of raw reads was done using FastQC to check the quality of raw reads.
Raw reads were sub-set by using seqtk sample to 22300000 millions reads per sample
Trimmomatic was used to trim the raw reads. (java -jar trimmomatic-0.32.jar PE -threads 4 -phred33 ILLUMINACLIP:adapters/TruSeq3-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:20 CROP:70 HEADCROP:5)
BWA was used to map the trimmed reads with the following parameters( bwa aln -n 2 -l 32 -k2 -t 8) followed by (bwa sampe -n1)
The ouput mapped files were converted to bam files using samtools view -Sb and samtools sort.
Unmapped reads were obtained using samtools view (-f 4) from the bam file obtained after mapping the samples to mm9. Then they were converted to bam files and from bam files later conterted to fastq file with bamToFastq (default)
the mapping to the Drosophila genome (dm39 was performed with the same parameters than used for mm9 mapping.
Quantification of the PRO-seq libraries was performed by using analyzeRepeats.pl rna mm9 -count genes -d for the coing genes and for the ncRNAs, using analyzeRepeats.pl NONCODEv4.gtf mm9 -count genes -d
The mapped files in bed format were used to create a Taq library using makeTagDirectory (-unique -sspe -mapq30).
The makeUCSCfile command (norm to spiking, (mapped reads on the drosophila genome (dm3)
genome build: mm9
processed data files format and content: Bedgraph_UCSC files to load in the UCSC genome Browser
|
| |
|
| Submission date |
Nov 14, 2017 |
| 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 |
GPL19057 |
| Series (1) |
| GSE76248 |
MiCEE: a ncRNA-protein complex mediates epigenetic silencing and nucleolus organization |
|
| Relations |
| BioSample |
SAMN08025900 |
| SRA |
SRX3390995 |
