|
| Status |
Public on Jun 25, 2010 |
| Title |
Cord blood CD4+_Act+IL-4_1h |
| Sample type |
SRA |
| |
|
| Source name |
human cord blood CD4+ cell, activated and IL-4 treated, 1h
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: cord blood
cell type: CD4+ T cells
|
| Treatment protocol |
Cells were activated through the T cell receptor (plate bound antiCD3 500 ng/24-well or proportially scaled up or down according to culturing well dimension, and soluble antiCD28 500 ng/ml, Immunotech, France), and to half of the samples IL-4 was added (10ng/ml, R&D Systems). Cells were cultured in Yssel´s medium. Cells were harvested at 1 and 4 h timepoints. Naive cells without any treatment were collected as control.
|
| Growth protocol |
Umbilical cord blood was collected from healthy neonates born in Turku University Hospital, Hospital District of Southwest Finland. Mononuclear cells were isolated with Ficoll-Paque (Amersham Biosciences) gradient centrifugation, and CD4+ cell population was purified using magnetic beads (Dynal CD4 Positive Isolation Kit, Invitrogen) according to manufacturer's instructions.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed using formaldehyde solution, lysed and sonicated to obtain chromatin fragments of 100-500bp size. The sonicated chromatin was then subjected to immunoprecipitation with polyclonal anti-STAT6 antibody (rabbit polyclonal αSTAT6 M-20, Santa Cruz Biotechnology, Inc.). The STAT6 bound chromatin was isolated using Dynal magnetic beads coated with secondary antibody anti-rabbit IgG (Dynal Biotech). The immunoprecipitated DNA was eluted, reverse crosslinked and purified using Qiagen minielute columns. The sample preparation for Solexa sequencing was performed according to Illumina's recommendations by Illumina service provider, Fasteris Life Sciences, Switzerland. Briefly, the ChIP DNA was end-repaired with T4 DNA polymerase, Klenow and PNK, and after purification with Qiagen minielute columns, a 3´-A was added using Klenow (exo-). The -A tailed DNA samples were purified and ligated with Illumina paired ends adaptors. After adapter ligation, fragments of size range of 200-300bp were purified from agarose gel and subjected to PCR amplification using Phusion polymerase with paired ends primers for 18 cycles. The libraries were quantified using PicoGreen Assay (Q-bit Invitrogen) before in situ amplification on the Illumina Cluster Station to generate DNA colonies according to the manufacturer's standard recommended protocol. Sequencing was performed on an Illumina Genome Analyzer “GAII” for 36 cycles using sequencing kit V-1.0 with scanning buffers V-2.0 and GA data analysis pipeline 1.0rc4. Each library was sequenced in a single read channel that produced between 4 to 5.2 million reads.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
Chromatin IP against STAT6
|
| Data processing |
Sequencing was performed on an Illumina Genome Analyzer and 27bp reads were aligned to the human reference genome (NCBI v36) using the short oligonucleotide alignment program SOAP (Li et al., Bioinformatics 24, 2008), allowing at most two mismatches. Only uniquely mapped reads were retained.
|
| |
|
| Submission date |
Aug 27, 2009 |
| Last update date |
May 15, 2019 |
| Contact name |
Henna Kallionpää |
| E-mail(s) |
[email protected]
|
| Phone |
+358-2-333-8001
|
| Organization name |
University of Turku
|
| Department |
Turku Centre for Biotechnology
|
| Lab |
Riitta Lahesmaa
|
| Street address |
P.O. Box 123
|
| City |
Turku |
| ZIP/Postal code |
FIN-20521 |
| Country |
Finland |
| |
|
| Platform ID |
GPL9115 |
| Series (2) |
| GSE17850 |
Genome-wide detection of STAT6 binding sites in IL-4 treated naive human CD4+ T cells |
| GSE18017 |
Stat6 mediated regulation of transcription to initiate Th2 program in human T cells |
|
| Relations |
| SRA |
SRX019003 |
| BioSample |
SAMN00011107 |
