|
| Status |
Public on Mar 17, 2023 |
| Title |
CCR6int_DB_A6393 |
| Sample type |
genomic |
| |
|
| Source name |
Healthy Donor PBMC
|
| Organism |
Homo sapiens |
| Characteristics |
array_id: Hsma-6229050122-R05C02
disease state: Healthy
cell type: PBMC
biosample: A6393
donor: D1
cell culture: day0
t-cell subset: CCR6int
|
| Treatment protocol |
DNA isolated immediately after sorting
|
| Growth protocol |
CD4+ T cells were isolated from elutriated lymphocytes by negative selection using RosetteSep CD4+ T cell enrichment cocktail (StemCell Technologies) followed by Ficoll/Hypaque (Amersham Biosciences) density centrifugation according to the protocol from StemCell Technologies. If necessary, red blood cells were removed using Pharm Lyse (BD Biosciences) according to the manufacturer’s protocol. For staining cells were suspended in 250 μl of Hanks Balanced Salt Solution (HBSS, Mediatech) containing 4% fetal bovine serum (FBS, GeminiBio) and incubated with following antibodies: anti-CD4, anti-CD45RO, and anti-CCR6 for 30 min at room temperature. In some experiments, in addition to these antibodies, cells were incubated with anti-CD25 and anti-CD62L, anti-CXCR5, anti-CXCR3 and anti-CCR4 antibodies. All antibodies were against human antigens. Cells were washed and resuspended in HBSS plus 4% FBS. CD4+ T cells were sorted into CD45RO- (naïve), and CD45RO+ subsets, and the CD45RO+ subset was further separated into CCR6neg, CCR6pos, and, within the CCR6pos cells, into CCR6low, CCR6int and CCR6high subsets. For in vitro polarization experiments, cells were sorted into naïve (CD4+CD45RO-CD25-CXCR5-CCR4-CXCR3-), CCR6neg (CD4+CD45RO+CD25-CXCR5-CXCR3-CCR6- or CD4+CD45RO+CD25-CXCR5- CCR4-CCR6- or CD4+CD45RO+CD25-CXCR5-CXCR3-CCR4-CCR6-), CCR6low (CD4+CD45RO+CD25-CXCR5-CXCR3-CCR6low or CD4+CD45RO+CD25-CXCR5-CCR4-CCR6low or CD4+CD45RO+CD25-CXCR5-CXCR3-CCR4-CCR6low), and CCR6high (CD4+CD45RO+CD25-CXCR5-CXCR3-CCR6high or CD4+CD45RO+CD25-CXCR5-CCR4-CCR6high or CD4+CD45RO+CD25-CXCR5-CXCR3-CCR4-CCR6high). All cell sorting was done using a FACSAria Cell Sorter (BD Biosciences). By post-sort analysis, the purity of populations was 95-99%. Cells were used for experiments immediately after sorting.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNeasy kit (Qiagen).
|
| Label |
biotin
|
| Label protocol |
Bisulfite conversion of genomic DNA (500 ng) was performed using the Zymo Research EZ DNA Methylation kit (Zymo Research) according to the manufacturer’s recommendations for the Illumina Infinium assay. The bisulfite conversion incubation was processed using alternative thermocycler settings as outlined in the “Alternative Incubation Conditions When Using the Illumina Infinium® Methylation” section of the Zymo instruction manual appendix.
|
| |
|
| Hybridization protocol |
bisulphite converted DNA was amplified, fragmented and hybridised to Illumina HumanMethylation450_15017482_v.1.2 using standard Illumina protocol
|
| Scan protocol |
Illumina HiSeq using standard protoocol
|
| Data processing |
Genome Studio 2011.1
Average Beta (normalized using Genome Studio 2011.1)
|
| |
|
| Submission date |
Mar 16, 2023 |
| Last update date |
Mar 17, 2023 |
| Contact name |
Timothy G Myers |
| E-mail(s) |
tgm@nih.gov
|
| Organization name |
National Institute of Allergy and Infectious Diseases
|
| Department |
Research Technologies Branch
|
| Lab |
Genomic Technologies Section
|
| Street address |
50 South Drive, Room 5509
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892-8005 |
| Country |
USA |
| |
|
| Platform ID |
GPL13534 |
| Series (2) |
| GSE227485 |
Human CCR6+ Th cells show both an extended stable gradient of Th17 activity and imprinted plasticity [humanmethylation450] |
| GSE227488 |
Human CCR6+ Th cells show both an extended stable gradient of Th17 activity and imprinted plasticity |
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