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Sample GSM8241114 Query DataSets for GSM8241114
Status Public on Jun 30, 2024
Title UT_Jurkat_MOZ_rep1
Sample type RNA
 
Source name Jurkat cells
Organism Homo sapiens
Characteristics treatment: untreated
target protein: MOZ
primary antibody: PA5-66742
primary antibody concentration: 20ug/ml
secondary antibody: Goat anti-rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (A32733)
secondary antibody concentration: 20ug/ml
Treatment protocol 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
 
Submission date Apr 29, 2024
Last update date Jun 30, 2024
Contact name Melissa McCalley Inge
Organization name Boston University
Department Biology
Lab Siggers
Street address 24 Cummington Mall
City Boston
State/province Massachusetts
ZIP/Postal code 02215
Country USA
 
Platform ID GPL34424
Series (2)
GSE266126 Rapid profiling of transcription factor-cofactor interaction networks reveals principles of epigenetic regulation (microarray)
GSE266127 Rapid profiling of transcription factor-cofactor interaction networks reveals principles of epigenetic regulation

Data table header descriptions
ID_REF
VALUE Processed data corresponds to z-scores normalized against the fluorescence distribution obtained for randomly selected background probes

Data table
ID_REF VALUE
hTF_v01_BG-1-chr14-84301932-84301965_NA_NA_NA_o1_r1 0.411176877
hTF_v01_BG-10-chr12-87918224-87918257_NA_NA_NA_o1_r1 -0.12115315
hTF_v01_BG-100-chr8-114271417-114271450_NA_NA_NA_o1_r1 0.501678161
hTF_v01_BG-101-chr7-58031636-58031669_NA_NA_NA_o1_r1 -0.04677743
hTF_v01_BG-102-chr1-241208962-241208995_NA_NA_NA_o1_r1 -0.616447814
hTF_v01_BG-103-chr4-10201662-10201695_NA_NA_NA_o1_r1 0.273167901
hTF_v01_BG-104-chr6-71713433-71713466_NA_NA_NA_o1_r1 -0.359555115
hTF_v01_BG-105-chr10-37877656-37877689_NA_NA_NA_o1_r1 -0.018443265
hTF_v01_BG-106-chr5-75221085-75221118_NA_NA_NA_o1_r1 0.39129771
hTF_v01_BG-107-chr5-17102844-17102877_NA_NA_NA_o1_r1 1.043779662
hTF_v01_BG-108-chr19-58546463-58546496_NA_NA_NA_o1_r1 -1.009182648
hTF_v01_BG-109-chr6-108803017-108803050_NA_NA_NA_o1_r1 -0.695991075
hTF_v01_BG-11-chr1-230576441-230576474_NA_NA_NA_o1_r1 -1.125528548
hTF_v01_BG-110-chr10-61262268-61262301_NA_NA_NA_o1_r1 -1.016348373
hTF_v01_BG-111-chr6-120276749-120276782_NA_NA_NA_o1_r1 -0.912410141
hTF_v01_BG-112-chr13-105290537-105290570_NA_NA_NA_o1_r1 0.299336614
hTF_v01_BG-113-chr3-7405896-7405929_NA_NA_NA_o1_r1 -0.26617585
hTF_v01_BG-114-chr1-52571052-52571085_NA_NA_NA_o1_r1 0.209865019
hTF_v01_BG-115-chr16-23653452-23653485_NA_NA_NA_o1_r1 -0.816159729
hTF_v01_BG-116-chr2-113041755-113041788_NA_NA_NA_o1_r1 -0.94464885

Total number of rows: 17434

Table truncated, full table size 826 Kbytes.




Supplementary file Size Download File type/resource
GSM8241114_UT_Jurkat_MOZ_rep1_647_p50_g550_3-4.gpr.gz 9.9 Mb (ftp)(http) GPR
GSM8241114_UT_Jurkat_MOZ_rep1_647_p50_g600_3-4.gpr.gz 10.3 Mb (ftp)(http) GPR
GSM8241114_UT_Jurkat_MOZ_rep1_647_p50_g700_3-4.gpr.gz 10.7 Mb (ftp)(http) GPR
GSM8241114_UT_Jurkat_MOZ_rep1_647_p50_g800_3-4.gpr.gz 11.2 Mb (ftp)(http) GPR
Raw data provided as supplementary file
Processed data included within Sample table

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