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| Status |
Public on Oct 19, 2021 |
| Title |
Carrier Treated Healthy Donor IgG CUT&RUN 72hr |
| Sample type |
SRA |
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| Source name |
Carrier Treated Healthy Donor IgG CUT&RUN
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| Organism |
Homo sapiens |
| Characteristics |
bach2 genotype: Wildtype (Healthy Donor)
cell type: Isolated and Treated Memory CD4+ Cells (Negative Magnetic Selection)
treatment: anti-CD3 and anti-CD28 Dynabeads, Carrier (Ethanol)
sequencing method: PE 50bp CUT&RUN, Novaseq
molecule subtype: CUT&RUN Fragment gDNA
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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 |
H3K27Ac and c-JUN CUT&RUN: CUT&RUN sequencing was performed on memory CD4+ T cells activated as above in the presence of carrier or VitD for 0.75h, 48h and 72h using the published protocol of Skene et al (Nature Protocols 2018). Antibodies against H3K27Ac (ab4729, Abcam), c-JUN (60AB, Cell Signaling Technologies), and non-specific IgG (31235, Thermo Fisher Scientific) were used with pAG-MNase (123461, Addgene) on 50,000 cells per target. CUT&RUN (and CUT&Tag below) buffer components were purchased from Sigma-Aldrich and Thermo Fisher Scientific. Post-CUT&RUN, short DNA fragments were prepared for paired-end sequencing (NEB Ultra II, New England Biolabs) with NovaSeq (Illumina). Amplified libraries were quantified by high sensitivity fluorometry (DeNovix) and sized via the 4200 TapeStation (Agilent Technologies).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Mem_CD4_Carrier.Treated.Healthy.Donor.IgG.CUTnRUN.72hr
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| Data processing |
H3K27Ac reads aligned to the human reference genome (GRCh37; hg19) using Bowtie2 with parameters ‘--local --maxins 250’ with sorting and indexing of aligned reads by samtools/1.10, reads with mapping quality <30 removed. H3K27Ac peaks were called by MACS2 with parameter ‘-f BAMPE --nomodel’. JUN reads were mapped without ‘--local’ parameter. <120 bp fragments were further sorted and indexed by samtools. JUN induced peaks called by MACS2 parameters ‘--nomodel -g hs -f BAMPE -q 0.01 --SPMR --keep-dup all’. IgG BAM files were included as controls for MACS2. Peaks for H3K27Ac and JUN experiments were screened against previously characterized hypersequencable-regions (Hg19 Blacklist, CutRunTools). H3K27ac and JUN mean binding intensities in peak regions calculated by UCSC bigWigAverageOverBed. Peaks, combined from two conditions, with mean binding intensity greater than 0.2 in at least one condition and fold change of signal>1.5 were differential. Known motifs +/−100bp from differential peak summits were identified (HOMER, findMotifsGenome.pl, parameter ‘-size given’). De novo motif footprinting for c-JUN was performed (CutRunTools). Average cuts per bp proximal to JUN peaks was plotted as a histogram relative to IgG. CUT&RUN tracks and heatmaps were visualized using IGV (Broad Institute) and deepTools, respectively.
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| Submission date |
Jul 20, 2020 |
| 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 |
SAMN15582181 |
| SRA |
SRX8772688 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4678085_Mem_CD4_Carrier.Treated.Healthy.Donor.IgG.CUTnRUN.72hr.bw |
70.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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