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| Status |
Public on Oct 19, 2021 |
| Title |
BACH2 Carrier CUT&Tag 96hr |
| Sample type |
SRA |
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| Source name |
Isolated and Treated Memory CD4+ Cells (Negative Magnetic Selection)
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| Organism |
Homo sapiens |
| Characteristics |
sequencing method: PE 50bp CUT&Tag, Novaseq
treatment: anti-CD3 and anti-CD28 Dynabeads, Carrier (Ethanol)
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| Treatment protocol |
Memory CD4 T Cell Isolation, Culture, and Treatment: Human PBMCs were purified from either anonymized leukodepletion cones (Blood Transfusion Service, NHS Blood and Transplantation) or healthy volunteer whole blood packs/buffy coats from the NIH Blood Bank and from fresh blood of patients. Leukodepletion cones were diluted 1 in 4 with sterile PBS and layered onto 15mL of lymphoprep (Axis-Shield). Whole blood packs/buffy coats were diluted 1 in 2 with sterile PBS onto 15mL Lymphocyte Separation Medium (25-072-CV LSM, Corning). Cells were separated by centrifugation at 20C for 20 min, with slow acceleration and no brakes and PBMC collected by harvesting interface cells. Human memory CD4+ T cells were used for RNA-seq, CUT&RUN, and CUT&Tag experiments as these cells express the VDR, while naïve T cells require pre-activation first. Human memory CD4+ T cells were isolated either from PBMCs using Miltenyi Memory CD4+ T cell Isolation Kit Human (130091893) or StemCell EasySep Human Memory CD4+ T Cell Enrichment Kit (19157) according to the manufacturer. Cells were cultured in Lonza X-VIVO-15 Serum-free Hematopoietic Cell Medium (04-418Q) supplemented with 50 IU/mL penicillin, 50µg/mL streptomycin (Invitrogen), 2mM L-glutamine (PAA Labarotories) at 37C 5% CO2. CD4+ memory T cells were cultured in a 96 well U bottom plate (Greiner) at a cell density of one million cells/mL in a total volume of 200µL. CD4+ cells were activated using Gibco Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation (11131D) at a cell to bead ratio of 4:1. Cells were additionally cultured in the presence or absence of 1α,25-Dihydroxyvitamin D3 (Enzo Life Sciences), reconstituted in 99.8% ethanol (Sigma-Aldrich), used at 10nM unless indicated. 99.8% ethanol was used as a carrier control at the same concentration.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
VDR, STAT3, and BACH2 CUT&Tag: VDR, STAT3, and BACH2 genome-wide binding in memory CD4+ T cells activated in the presence of 10nM VitD or carrier for 96h was detected by CUT&Tag as per the published protocol (CUT&Tag V.3, dx.doi.org/10.17504/protocols.io.bcuhiwt6) using fixed nuclei (2min fixation of isolated nuclei, RT) with 75,000 nuclei/target and antibodies targeting either VDR (D2K6W, Cell Signaling Technologies), STAT3 (D3Z2G, Cell Signaling Technologies), BACH2 (D3T3G, Cell Signaling Technologies), or non-specific IgG (31235, Thermo Fisher Scientific) diluted 1:50 accordingly and incubated with fixed-nuclei for 1h, RT. Secondary antibody (1:100) (ABIN101961, antibodies-online) was bound for 0.5h, RT. Preloaded pA-Tn5 (C01070001, Diagenode) was tethered to antibodies for 1h, RT. Libraries were sequenced paired-end by Novaseq.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Resulting reads were processed and mapped to Hg19 (CutRunTools), with peaks called (SEACR/v1.2, stringent, all mapped fragments) and hypersequencable-regions subtracted. Known motif enrichment was determined +/−200bp from peak summits and data visualized as in CUT&RUN.
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| Submission date |
Oct 15, 2021 |
| Last update date |
Oct 20, 2021 |
| Contact name |
Behdad (Ben) Afzali Khoshkbijari |
| E-mail(s) |
ben.afzali@nih.gov
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| Phone |
301-443-2055
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| Organization name |
National Institutes of Diabetes, Digestive, and Kidney Diseases
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| Department |
Kidney Diseases Branch
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| Lab |
Immunoregulation Section
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| Street address |
10 Center Drive, 10/8D03
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE154741 |
Autocrine vitamin D signaling switches off pro-inflammatory programs of Th1 cells |
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| Relations |
| BioSample |
SAMN22315379 |
| SRA |
SRX12629690 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5627776_BACH2.Carrier.bw |
5.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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