|
| Status |
Public on Feb 28, 2017 |
| Title |
STATegra Ikaros cells 24h Batch1 |
| Sample type |
SRA |
| |
|
| Source name |
pre-B lymphocyte
|
| Organism |
Mus musculus |
| Characteristics |
transfection: inducible Ikaros
time: 24h
#replicate: 1
#batch: 1
treatment: 4-OHT
dose: 0.5 uM
cell line: B3
cell type: pre-B lymphocyte
|
| Treatment protocol |
Cells containing inducible Ikaros were generated by transducing mouse pre-B cell line B3 with mouse stem cell virus (MSCV) retroviral vectors encoding a fusion protein of haemagglutinin-tagged wild type Ikaros (HA-Ikaros) and the estrogen receptor hormone-binding domain (ERt2), followed by an internal ribosomal entry site (IRES) and GFP. B3 cells containing inducible Ikaros (HA-Ikaros-ERt2-IRES-GFP) were plated at a density of 0.5 million cells/ml in IMDM medium containing 10% FCS and 1% penicillin-streptomycin. 0.5uM 4 hydroxy-tamoxifen was added to the cells 2h before collection.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
B3 cells containing inducible Ikaros were plated at a density of 0.5 million cells/ml in IMDM medium supplemented with 10% FCS and 1% penicillin/streptomycin. Time point samples were collected by 5 min centrifugation at 1200 rpm. Cell pellets were washed 2 times in PBS, frozen in liquid nitrogen and storaged at -80. Control vector-ERt2 B3 cells were plated at a density of 0.5 million cells/ml in IMDM medium supplemented with 10% FCS and 1% penicillin/streptomycin. Samples were collected by 5 min centrifugation at 1200 rpm. Cell pellets were washed 2 times in PBS, frozen in liquid nitrogen and storaged at -80.
Small-RNA Seq libraries were generated using TruSeq Small RNA Sample Preparation Kit following the manufacter’s instructions. In brief, the kit takes advantage of the hallmark 3’ hydroxyl group microRNAs have that result from the enzymatic cleavage by Dicer (and other RNA processing enzymes) and an adenylated 3’ adapter that specifically ligates to this end. By targeting the 3’ end of small RNAs, unwanted ligation to other transcripts is reduced allowing for specific sequencing of microRNAs and other 3’OH transcript species. The protocol involves: isolation of total RNA; ligation of the 3’ adapter; addition of the hairpin oligo to reduce adapter dimers; ligation of the 5’ adapter; annealing of the primer to the 3’ adapter and reverse transcription and PCR amplification and barcode addition.
|
| |
|
| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
24h after Taxomifen induction in Ikaros/ERt2 cells
|
| Data processing |
miRNA-seq data were trimmed and aligned to the mm10 reference (source miRbase) using Novoalign software
Counts were obtained using multiBamCov (Bedtools suite)
Prior to normalize the data, low count miRNAS were filtered out with CPM (counts per million) method in NOISeq R package
TMM method was used to normalize the data
ComBat was used to reduce batch effect
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files containing smallRNA quantification values for each Sample (matrix).
|
| |
|
| Submission date |
Nov 25, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Ana Conesa |
| E-mail(s) |
info@stategra.eu
|
| Organization name |
Centro de Investigaciones Príncipe Felipe (CIPF)
|
| Department |
Computational Genomics Program
|
| Lab |
Genomics and Gene Expression Lab
|
| Street address |
C/ Eduardo Primo Yúfera 3
|
| City |
Valencia |
| State/province |
Valencia |
| ZIP/Postal code |
46012 |
| Country |
Spain |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE75394 |
miRNA-seq analysis of B3 pre-B cell line from STATegra Project |
| GSE75395 |
Omics analyses of B3 pre-B cell line from STATegra Project |
|
| Relations |
| BioSample |
SAMN04295823 |
| SRA |
SRX1452184 |
