Jurkat cells intended to be stimulated as well as paired untreated cells were plated at 1 x 106 cell/ml 2 hours prior to stimulation. To stimulate selected cells, soluble anti-CD28, CD3 & CD2 (ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator, Catalog #10970) was added to media at manufacturers recommended 25 ul/ml. Cells were agitated slightly and placed back in the incubator for 45 minutes. After 45 minutes, both the untreated and stimulated cells were processed for nuclear.
Growth protocol
SUDHL4 (CRL-2957), Jurkat E6-1 (TIB-152), and HEK293 (CRL-1573) cells were obtained from ATCC. SUDHL4 and Jurkat cells were grown in suspension RPMI 1640 Glutamax media (Thermofisher Scientific, Catalog #72400120) with 10% heat-inactivated fetal bovine serum (Thermofisher Scientific, Catalog #132903) and Jurkat cells medium was also supplemented with 1mM sodium pyruvate (Thermofisher Scientific, Catalog #16140071). Cells were grown at 37 °C with 5% CO2. T175 (CELLTREAT, Catalog #) non-treated flasks were used when culturing SUDHL4 and Jurkats cells for experiments. HEK293 cells were grown in low glucose DMEM (Cytiva, Catalog # SH30021) supplemented with 10% FBS at 37 °C with 5% CO2. HEK293 cells were grown in cell culture treated T225 flasks (CELLTREAT, catalog #) for experiments.
Extracted molecule
total RNA
Extraction protocol
Nuclear extracts were obtained as previously described (Bray et al. PMID: 35252945, Mohaghegh et al. PMID: 30657937) with modifications detailed below. To harvest nuclear extracts from Jurkat cells, the cells were collected in a 15 ml falcon tube and placed on ice. To harvest nuclear extracts from SK-MEL-28 cells, the media was aspirated off and the cells were washed once with 1X PBS (Thermofisher Scientific, Catalog #100010049). Once the 1X PBS used to wash the cells was aspirated off, enough 1X PBS was mixed with 0.1mM Protease Inhibitor (Sigma-Aldrich, Catalogue #P8340) to cover the cells was added to each flask. A cell scraper was used to dislodge the cells from the flask, and cells were collected in a 15 ml falcon tube and placed on ice. Jurkat and SK-MEL28 cells were pelleted by centrifugation at 500xg for 5 min at 4˚C. Both pellets were washed with 2mL of 1X PBS with Protease Inhibitor and pelleted again at 500xg for 2 min at 4˚C. To lyse the plasma membrane, the cells were resuspended in Buffer A (1 mL Buffer A for Jurkat cells, 1.5 mL Buffer A for SK-MEL28 cells) (10mM HEPES, pH 7.9, 1.5mM MgCl, 10mM KCl, 0.1mM Protease Inhibitor, Phosphatase Inhibitor (Santa-Cruz Biotechnology, Catalog #sc-45044), 0.5mM DTT (Sigma-Aldrich, Catalog #4315) and incubated for 10 min on ice. After the 10 min incubation, Igepal detergent (final concentration of 0.1%) was added to the cell and Buffer A mixture and vortexed for 10 s. To separate the cytosolic fraction from the nuclei, the sample was centrifuged at 500xg for 5 min at 4˚C to pellet the nuclei. The cytosolic fraction was collected into a separate microcentrifuge tube. The pelleted nuclei were then resuspended in Buffer C (100 µL for Jurkat nuclei and 150 µL for SK-MEL-28 nuclei) (20mM HEPES, pH 7.9, 25% glycerol, 1.5mM MgCl, 0.2mM EDTA, 0.1mM Protease Inhibitor, Phosphatase Inhibitor, 0.5mM DTT, and 420mM NaCl) and then vortexed for 30 s. To extract the nuclear proteins (i.e., the nuclear extract), the nuclei were incubated in Buffer C for 1 h while mixing at 4˚C. To separate the nuclear extract from the nuclear debris, the mixture was centrifuged at 21,000xg for 20 min at 4˚C. The nuclear extract was collected in a separate microcentrifuge tube and flash frozen using liquid nitrogen. Nuclear extracts were stored at -80˚C
Label
Alexa fluor conjugated antibody
Label protocol
PBM experiments using cell extracts were performed following the protocols previously described (Berger et al. PMID: 19265799, Mohaghegh et al. PMID: 30657937) and outlined here. The double-stranded microarray was pre-wetted in HBS+TX-100 (20mM HEPES, 150mM NaCl, 0.01% Triton X-100) for 5 min and then de-wetted in an HBS bath. Each of the microarray chambers were then incubated with 180 mL of nuclear extract binding mixture for 1 h in the dark. Nuclear extract binding mixture (per chamber): 400-600 mg of nuclear extract; 20mM HEPES (pH 7.9); 100mM NaCl; 1mM DTT; 0.2mg/mL BSA; 0.02% Triton X-100; 0.4mg/mL salmon testes DNA (Sigma-Aldrich, Catalog #D7656)). The microarray was then rinsed in an HBS bath containing 0.1% Tween-20 and subsequently de-wetted in an HBS bath. After the nuclear extract incubation, the microarray was incubated for 20 min in the dark with 20mg/mL primary antibody for the TF or COF of interest. The primary antibody was diluted in 180 mL of 2% milk in HBS. The array was then rinsed in an HBS bath containing 0.1% Tween-20, de-wetted in an HBS bath, and incubated with 10mg/mL of either Alexa488- or Alexa647-conjugated secondary antibody for 20 min in the dark. The secondary antibody was diluted in 180 mL of 2% milk in HBS. Excess antibody was removed by washing the array twice for 3 min in 0.05% Tween-20 in HBS and once for 2 min in HBS in Coplin jars as described above. After the washes, the microarray was de-wetted in an HBS bath and scanned
Hybridization protocol
Microarray DNA double stranding and PBM protocols are as previously described (Berger et al. PMID: 19265799, Mohaghegh et al. PMID: 30657937)
Scan protocol
Microarrays were scanned with a GenePix 4400A scanner and fluorescence was quantified using GenePix Pro 7.2
Data processing
Exported fluorescence data from GenePix Pro 7.2 were normalized with MicroArrary LINEar Regression (Berger et al, 2006). Processed data corresponds to z-scores normalized against the fluorescence distribution obtained at sets of random background probes
