TeloHAEC basal rep2
Homo sapiens
Protocols: Chromatin was isolated from 5 x 10^6 TeloHAEC cells in 1 mL of 1X NUN buffer (20 mM HEPES, 7.5 mM MgCl2, 0.2 mM EDTA, 0.3 M NaCl, 1M urea, 1% NP-40, 1 mM DTT, 20 units/mL SUPERase In RNase Inhibitor (Thermo Fisher Scientific, Waltham, MA, AM2694), 1X Protease Inhibitor Cocktail (Roche, 11836145001)). Samples were vortexed for 1 minute, an additional 500 µL of 1x NUN buffer was added to each sample and were vortexed again for another 30 seconds. Samples were incubated in Thermomixer (Eppendorf, Hamburg, Germany) at 4°C with shaking at 1500 rpm for 30 minutes followed by centrifugation at 12,500 x g for 30 minutes at 4°C. Each sample was washed thrice with 1 mL 50 mM Tris-HCl (pH 7.5) supplemented with 40 units/mL SUPERase In RNase Inhibitor and centrifuged at 10,000 x g for 5 minutes at 4°C. 100 µL of chromatin storage buffer (50 mM Tris-HCl pH 8.0, 25% glycerol, 5 mM magnesium acetate, 0.1 mM EDTA, 5 mM DTT, and 40 units/mL SUPERase In RNase Inhibitor) was added to the chromatin pellet and incubated on ice for 5mins. Samples were placed in a Bioruptor pico sonicator (Diagenode, Denville, NJ) and sonicated for 10 cycles of 30 seconds ON and 30 seconds OFF. Sonication was repeated for another two times to completely solubilize the chromatin and samples were snap frozen on liquid nitrogen and stored at -80°C. To induce the acute pro-inflammatory response, TeloHAECs (n=2; two biological replicates) were treated with 10 ng/mL recombinant human TNFα (Cell Applications, cat# RP1111 - 50). The un-stimulated control samples were treated with vehicle ( i.e. an equivalent amount of water in MV2 media without supplements). 100 µL of solubilized chromatin was mixed with 100 uL 2X run-on reaction mix (10 mM Tris-HCl pH 8.0, 5 mM MgCl2, 1 mM DTT, 300 mM KCl, 400 µM ATP, 400 µM GTP, 40 µM Biotin-11-UTP (Perkin Elmer, Waltham, MA, NEL543001EA), 40 µM Biotin-11-CTP (Perkin Elmer, Waltham, MA, NEL542001EA), 0.8 units/µL SUPERase In RNase Inhibitor, 1% sarkosyl). The run-on reaction was performed at 37°C for 5 minutes and stopped by adding 500 µL Trizol LS (Thermo Fisher Scientific, 10296-010) to the reaction. RNA samples were precipitated with ethanol with glycoblue as coprecipitant and resuspended in 20ul of diethylpyrocarbonate (DEPC) treated water and heat denatured at 65°C for 40 seconds, and base hydrolysis was performed with 0.2N NaOH on ice for 8 minutes. Nascent RNA was pulled-down with streptavidin beads (New England Biolabs (NEB), Ipswich, MA, S1421S). RNA was extracted using Trizol (Thermo Fisher Scientific, 15596-026) 3' adaptor ligation was performed with T4 RNA Ligase 1 (NEB, M0204L). Second pull down with streptavidin beads was performed followed by 5' decapping with RNA 5' pyrophosphohydrolase (RppH, NEB M0356S). The 5' end of the RNA molecule was phosphorylated with T4 polynucleotide kinase (PNK, NEB M0201S) and 5' adaptor ligation was performed with T4 RNA Ligase 1. Third streptavidin bead pulldown was performed again followed by reverse transcription using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, 18080-044). cDNA was amplified by PCR using the Q5 High-Fidelity DNA Polymerase (NEB, M0491S) to generate ChRO-seq libraries.
ENA-FIRST-PUBLIC
2020-08-20T04:04:39Z
ENA-LAST-UPDATE
2020-08-10T15:58:46Z
External Id
SAMEA7171710
INSDC center name
University of Toronto The Hospital for Sick Children
INSDC first public
2020-08-20T04:04:39Z
INSDC last update
2020-08-10T15:58:46Z
INSDC status
public
Submitter Id
E-MTAB-9425:TeloHAEC basal rep2
broker name
ArrayExpress
cell line
TeloHAEC
cell type
endothelial cell
common name
human
developmental stage
adult
disease
normal
organism part
aorta
replicate
2
sample name
E-MTAB-9425:TeloHAEC basal rep2
scientific_name
Homo sapiens
sex
female
stimulus
basal