GSM5627775: BACH2 VitD CUT&Tag 96hr; Homo sapiens; OTHER
Nextera 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 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 manufacturers' instructions.    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 at a cell density of 106 cells per 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) or 25-Dihydroxyvitamin D3 (Enzo Life Sciences), both reconstituted in 99.8% ethanol (Sigma-Aldrich), used at 10nM unless indicated. 99.8% ethanol was used as a carrier control at the same concentrations. VDR, STAT3, and BACH2 CUT&Tag: To detect genome-wide binding of VDR, STAT3, and BACH2 in Memory CD4+ cells activated with anti-CD3+anti-CD28 in the presence of 10nM VitD or carrier for 96 hr, CUT&Tag sequencing was performed as per the published protocol (CUT&Tag V.3, dx.doi.org/10.17504/protocols.io.bcuhiwt6) (Kaya-Okur et al. 2019) using the fixed nuclei module (2min fixation of isolated nuclei at RT) with 75,000 nuclei per CUT&Tag reaction with only the following specifications. CUT&Tag was performed with rabbit anti- VDR (D2K6W, Cell Signaling Technologies), STAT3 (D3Z2G, Cell Signaling Technologies), BACH2 (D3T3G, Cell Signaling Technologies), and non-specific IgG (31235, Thermo Fisher Scientific) antibodies (1:50) for 1 hr at RT, followed by washing and guinea pig anti-rabbit IgG (ABIN101961, antibodies-online) secondary antibody (1:100) binding for 30 min. Recombinant pA-Tn5 fusion protein was obtained pre-loaded with sequencing adapters (C01070001, Diagenode) and bound to antibodies for 1 hr at RT. Normalized sequencing libraries were sequenced via NovaSeq (Illumina). Resulting reads were processed by CutRunTools and mapped to the human reference (Hg19) with peaks called by SEACR (Sparse Enrichment Analysis for CUT&RUN, https://github.com/FredHutch/SEACR) with stringent settings including all mapped fragment sizes. Hypersequencable regions and IgG peaks were subtracted from resulting peaks. Known motif finding on identified peaks (+/- 200bp from peak summits) was performed via Homer findMotifsGenome.pl using parameter '-size given'. CUT&Tag tracks and heatmaps were visualized using IGV browser (Broad Institute) and deepTools, respectively.