|
| Status |
Public on Jun 15, 2018 |
| Title |
E6Untreated1 |
| Sample type |
SRA |
| |
|
| Source name |
Aire KO_mTEc cells_without co-culture
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell line: mTEC 3.10
cell type: medullary thymic epithelial (mTEC)
passages: 2
genotype: Aire +/-
|
| Treatment protocol |
Thymus of C57BL/6 mice was surgically removed, minced and fragments filtered through a nylon membrane to separate the thymocytes. The thymocytes were co-cultured with Aire KO or WT mTEC cells in a ratio 50:1 (thymocytes:mTECs) for 12h. The adherent thymocytes were separated by washing with PBS. The mTECs cells were trypsinized and collected for further total RNA extraction.
|
| Growth protocol |
1x10^6 Aire KO or WT mTEC 3.10 cells were cultured in RPMI 1640 medium (37 °C with 5% CO2) for 24h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
mirVana miRNA Isolation Kit (Ambion) was used for total RNA extraction from Aire KO mTEC 3.10 cells (clone E6) according to the manufacturer's instructions. The RNA samples were eluted in RNAse free water and stored in a -80 C freezer. The yield was determined by spectrophotometry (A260) and analyzed for phenol or protein contamination, (A260/A230) and (A260/A280), respectively. The integrity of RNA samples was evaluated by microfluidic electrophoresis using Agilent RNA Nano 6000 chips and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). (Parameters: Extracted product = total_RNA, Amplification = none)
RNA libraries were prepared for sequencing using standard Illumina protocol to the TruSeq Stranded mRNA Library Preparation kit (RS-122-2101).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Illumina Casava1.7 software used for basecalling
Sequenced reads were trimmed for adaptor sequence, and masked for low-quality sequence using the FASTX-Toolkit v 0.0.14 (By Hannon Lab - http://hannonlab.cshl.edu/fastx_toolkit/index.html), then mapped to mm10 whole genome using the splice-aware aligner STAR v.2.5.0a along with the UCSC annotation file (gtf) for GRCm38/mm10 (Dec.2011).
Gene counts were compiled using the HTseq tool. Only genes that had at least 20 reads in any given library were used in subsequent analysis.
Normalization and differential expression was carried out using the DESeq2 Bioconductor package with the R statistical programming environment.
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited text files containing Gene Name, Number of reads (raw counts by HTSesq-counts)
|
| |
|
| Submission date |
Dec 07, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Geraldo A Passos |
| Organization name |
University of Sao Paulo
|
| Department |
Genetics
|
| Lab |
Molecular Immunogenetics
|
| Street address |
3900, Bandeirantes Avenue
|
| City |
Ribeirao Preto |
| State/province |
SP |
| ZIP/Postal code |
14049900 |
| Country |
Brazil |
| |
|
| Platform ID |
GPL17021 |
| Series (1) |
| GSE91015 |
Crispr-Cas9-mediated Aire gene editing in medullary thymic epithelial (mTEC) cells shows its role as a gene expression modulator during thymocyte adhesion |
|
| Relations |
| BioSample |
SAMN06118038 |
| SRA |
SRX2403401 |
