|
| Status |
Public on Nov 24, 2012 |
| Title |
RNAseq_WTTh2_Gata3V |
| Sample type |
SRA |
| |
|
| Source name |
primary CD4+ T cells from spleen and lymph nodes
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
genotype/variation: wild type
cell type: in vitro polarized T helper2 cells for 5 days with Gata3 overexpression
passages: Gata3 overexpression vector was made in Th2 cells.
|
| Treatment protocol |
To perform retroviral transduction of CD4+ T cells, sorted naïve CD4+ T cells from WT or Stat6-/- mice were cultured in the presence of plate-bound anti-CD3 and anti-CD28 (10 μg/mL each) with anti-IFNG (10 μg/mL) for 16 h. Culture medium was replaced with retroviral soup and 4 μg/ml polybrene, followed by centrifugation at 2500 rpm for 2 hours. After 4-hour incubation at 37 C, viral supernatant was replaced with Th2 cell culture medium containing IL-4 (10 ng/mL) and anti-IFNG for 2 days. After that, cells were released from TCR stimulation and were cultured further in IL-2 (50 U/mL) and IL-4 and grown an additional 3 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared from 2-5 million cells using mirVana miRNA Isolation kit (#AM1560, ABI). One microgram of total RNA was subsequently used to prepare RNA-seq library using TruSeq SRRNA sample prep kit (FC-122-1001,Illumina) by following manufacturer's protocol. The libraries were sequenced for 100 cycles (single read) with HiSeq 2000 (Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
RNA-seq
|
| Data processing |
For H3K4me1: the unique tags were mapped into non-overlapping 200 bp windows of the mouse genome. Significant islands were identified based on window tag-count threshold determined from a p-value=0.05 defined by Poisson background model using SICER, a method appropriate for broad peaks
For p300 and STATs CisGenome v2.0, an extension of the earlier version (Ji et al., 2008), was utilized based on the normal rabbit serum as the control IP. The parameters used for seq-peak command were c=2 or 3, b=100, and e=0.
RNA-seq: tophat and cufflinks. Sequence reads from each cDNA library were mapped onto the mouse genome build mm9 using tophat and the mappable data were then processed by Cufflinks (Trapnell et al.). The obtained data were normalized based on RPKM (reads per kilobase exon model per million mapped reads).
Genome_build: mm9
|
| |
|
| Submission date |
Aug 29, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Golnaz Vahedi |
| Organization name |
National Institutes of Health
|
| Department |
NIAMS
|
| Lab |
Lymphocyte Cell Biology Section
|
| Street address |
9000 Rockville Pike Bldg 10 Rm 13C101A
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE40463 |
STATs Shape the Active Enhancer Landscape of T Cell Populations |
|
| Relations |
| SRA |
SRX183973 |
| BioSample |
SAMN01162811 |
| Named Annotation |
GSM994547_WT_Th2_Gata3V.bigwig |
