|
| Status |
Public on Jun 22, 2010 |
| Title |
FOXP3Genome_hs18_7 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
FOXP3 ChIP DNA from expanded Human T regulatory cells
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: cord blood
cell type: CD4+CD25+ natural T regulatory cells (nTreg)
antibody: Rabbit polyclonal anti-human FOXP3 IgG (Novus Biologicals)
|
| Treatment protocol |
Expanded cord blood Treg cells were cultured overnight prior to re-stimulation (2hours) with 1mM Ionomycin (Sigma) before Formaldehyde cross-linking.
|
| Growth protocol |
Cord blood CD4+ CD25+ (nTreg) cells were isolated from MNC using a Dynabeads Regulatory CD4+CD25+ T cell kit (Invitrogen). The purity for each cell type was routinely greater than 90% by two colour flow cytometry for CD4 and CD25 expression. Ex vivo expansion of isolated T cell populations (1x106 cells/well in a 24 well plate) were performed in X-Vivo 15 media (Lonza) supplemented with 20mM HEPES-, pH 7.4, 5% heat inactivated pooled human serum (Lonza), 2mM l-glutamine and 500U/ml recombinant human interleukin-2 (rhIL2; R&D research) in the presence of Dynabeads CD3/CD28 T cell expander beads (Invitrogen; Cat# 111-41D) at a bead to cell ratio of 3:1. Cells were expanded for 8 days in the presence of Dynabeads prior to magnetic removal of the beads and culture in X-Vivo 15 media supplemented as above except with 100U/ml rhIL2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were treated for 10 minutes in 1% formaldehyde solution (50 mM HEPES KOH, pH 7.5, 100mM NaCl, 1mM EDTA, 0.5mM EGTA and 11% formaldehyde). Cell lysis, chromatin immunoprecipitation (ChIP) and DNA isolation steps for human FOXP3 ChIP-on-chip experiments were carried out essentially as described in (Lee et al. 2006; Nature Protocols, 1(2), 3599-3606), using 5x107 cells per immunoprecipitation with either a rabbit anti-Foxp3 IgG (Novus Biochem) or ChIP grade control rabbit IgG Sera (Abcam). Chromatin shearing conditions was performed with a Misonix S-4000 ultrasonic processor (Misonix) with a microtip. Total input chromatin was also purified from an aliquot (100μl) of crosslinked material and the concentration determined using a Nanodrop UV spectrophotometer (Thermo Scientific). Amplification of immunoprecipitated and input chromatin (10ng) for labelling and hybridisation to Affymetrix Human Tiling 2.0R arrays was by whole genome amplification (WGA; Sigma GenomePlex WGA kit) (O'Geen et al. 2006; Biotechniques 41(5) 577-580 ) adapted for Affymetrix labelling systems by the inclusion of 0.11mM dUTP in the amplification reaction. Amplified material was purified using a PCR clean up kit (QIAGEN). Labelling and hybridisation of amplified material was carried out at the Biomolecular Resource Facility (John Curtin School of Medical Research, Australian National University).
|
| Label |
biotin
|
| Label protocol |
Samples (~9μg of DNA) were enzymatically fragmented and labeled using the WT Labeling Kit (Affymetrix) to add biotinylated compound to the ends of the fragments
|
| |
|
| Channel 2 |
| Source name |
Input DNA from expanded Human T regulatory cells
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: cord blood
cells: CD4+CD25+ natural T regulatory cells (nTreg)
antibody: None
|
| Treatment protocol |
Expanded cord blood Treg cells were cultured overnight prior to re-stimulation (2hours) with 1mM Ionomycin (Sigma) before Formaldehyde cross-linking.
|
| Growth protocol |
Cord blood CD4+ CD25+ (nTreg) cells were isolated from MNC using a Dynabeads Regulatory CD4+CD25+ T cell kit (Invitrogen). The purity for each cell type was routinely greater than 90% by two colour flow cytometry for CD4 and CD25 expression. Ex vivo expansion of isolated T cell populations (1x106 cells/well in a 24 well plate) were performed in X-Vivo 15 media (Lonza) supplemented with 20mM HEPES-, pH 7.4, 5% heat inactivated pooled human serum (Lonza), 2mM l-glutamine and 500U/ml recombinant human interleukin-2 (rhIL2; R&D research) in the presence of Dynabeads CD3/CD28 T cell expander beads (Invitrogen; Cat# 111-41D) at a bead to cell ratio of 3:1. Cells were expanded for 8 days in the presence of Dynabeads prior to magnetic removal of the beads and culture in X-Vivo 15 media supplemented as above except with 100U/ml rhIL2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were treated for 10 minutes in 1% formaldehyde solution (50 mM HEPES KOH, pH 7.5, 100mM NaCl, 1mM EDTA, 0.5mM EGTA and 11% formaldehyde). Cell lysis, chromatin immunoprecipitation (ChIP) and DNA isolation steps for human FOXP3 ChIP-on-chip experiments were carried out essentially as described in (Lee et al. 2006; Nature Protocols, 1(2), 3599-3606), using 5x107 cells per immunoprecipitation with either a rabbit anti-Foxp3 IgG (Novus Biochem) or ChIP grade control rabbit IgG Sera (Abcam). Chromatin shearing conditions was performed with a Misonix S-4000 ultrasonic processor (Misonix) with a microtip. Total input chromatin was also purified from an aliquot (100μl) of crosslinked material and the concentration determined using a Nanodrop UV spectrophotometer (Thermo Scientific). Amplification of immunoprecipitated and input chromatin (10ng) for labelling and hybridisation to Affymetrix Human Tiling 2.0R arrays was by whole genome amplification (WGA; Sigma GenomePlex WGA kit) (O'Geen et al. 2006; Biotechniques 41(5) 577-580 ) adapted for Affymetrix labelling systems by the inclusion of 0.11mM dUTP in the amplification reaction. Amplified material was purified using a PCR clean up kit (QIAGEN). Labelling and hybridisation of amplified material was carried out at the Biomolecular Resource Facility (John Curtin School of Medical Research, Australian National University).
|
| Label |
biotin
|
| Label protocol |
Samples (~9μg of DNA) were enzymatically fragmented and labeled using the WT Labeling Kit (Affymetrix) to add biotinylated compound to the ends of the fragments
|
| |
|
| |
| Hybridization protocol |
Approximately 9 μg of DNA was hybridzed per array using the Affymetrix hybridization kit. Arrays were hybridized for 16 hours at 45° at 60rpm using an Affymetrix hybridization oven and washed with Fluidics station 450 protocol FS450_0001
|
| Scan protocol |
Arrays were scanned on an Affymetrix Scanner 3000 7G
|
| Description |
FOXP3 ChIP Biological Rep 1-2, chip G
|
| Data processing |
All analysis was performed with NCBI build 36/hg18 of the human genome. The model-based analysis of tiling array (MAT) algorithm (Johnson WE et al 2006; PNAS 103(33), 1257-12462) was used to find regions of FOXP3 binding. Matched total input DNA were used as control samples.
FOXP3 ChIP in primary human natural T regulatory cells, using Total Input DNA as control, 2 biological replicates. The supplementary 'Treg.ab.Hs.Tiling2.0.FDR0.5.bed' file contains 8305 high quality binding FOXP3 binding regions.
|
| |
|
| Submission date |
Mar 22, 2010 |
| Last update date |
Jun 22, 2010 |
| Contact name |
Stephen Martin Pederson |
| E-mail(s) |
stephen.pederson@adelaide.edu.au
|
| Phone |
+614 1333 9618
|
| Organization name |
University of Adelaide
|
| Department |
Biological Sciences
|
| Lab |
Boinformatics Hub
|
| Street address |
North Terrace
|
| City |
Adelaide |
| State/province |
SA |
| ZIP/Postal code |
5005 |
| Country |
Australia |
| |
|
| Platform ID |
GPL4916 |
| Series (1) |
| GSE20995 |
Genome wide FOXP3 binding sites in human cord blood derived CD4+CD25+ natural T regulatory cells (nTreg) |
|
