NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM2333689 Query DataSets for GSM2333689
Status Public on Aug 29, 2018
Title HUVEC_Scr_IL1beta_FA_Input
Sample type SRA
 
Source name Human umbilical vein endothelial cells (HUVECs)
Organism Homo sapiens
Characteristics tissue: Human umbilical vein endothelial cells (HUVECs)
passages: Passage 4
state/ treatment: Confluent, treated with Scr siRNA and stimulated with IL-1β
chip antibody: None
chip antibody lot # and amount used pr. chip-seq: None
Treatment protocol Untreated cells were cultured as described in the growth protocol section until crosslinking and harvest. For knockdown experiments, cells were transfected with 27 nM siRNA targeting Notch1 or negative control siRNA (Scr) using Lipofectamine RNAiMAX and 60 hours later stimulated with or without 5 ng/ml of IL-1β for 1 hour.
Growth protocol Pools of HUVECs from 13 donors were grown on 15 cm culture dishes coated with 0.1% gelatin in MCBD 131 medium supplemended with 7.5% fetal calf serum, 10 ng/ml EGF, 1 ng/ml bFGF, 1 μg/ml hyrdocortisone, 50 μg/ml gentamicin, 250 ng/ml fungizone, and 1% L-glutamine. HUVECs that reached compact, high-density monolyer. We define the superconfluent state as described earlier in Küchler et al. Am J Pathol. 2008 Oct;173(4):1229-42.
Extracted molecule genomic DNA
Extraction protocol For Notch1, RBPJ, and NF-κB/RelA ChIP-seq experiments, cells were crosslinked in a two-step crosslinking procedure: 45 min in 2 mM disuccinimidyl glutarate (DSG) followed by 10 min incubation with 1% formaldehyde (FA) before quenching with 125 mM glycine. For H3K27ac ChIP-seq, only FA crosslinking was applied. For each 15 cm dish, cells were lysed in 1.6 mL lysis buffer (0.1% SDS, 1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 20 mM Tris pH 8), removed with a cell scraper, and kept at 4°C before sonication (material used for NF-κB/RelA and H3K27ac ChIP-seq was stored at -80°C). DSG/FA and FA samples were sonicated for 40 and 16 cycles, respectively, on a Bioruptor Twin set to 30 sec ON/OFF and high intensity. To remove cell debris, samples were subjected to centrifugation at 10,000g for 1 min and supernatants were collected. ChIP was performed on approximately 1.5e7 cells in a final volume of 6 mL lysis buffer with BSA in 15 mL tubes. Chromatin was incubated with antibodies as indicated in the list above for 3 hours at 4°C with upside-down rotation. Protein A sepharose beads were added and samples were incubated overnight at 4°C with upside-down rotation. Beads were washed in 12 mL by the following wash steps: 2x wash buffer 1 (0.1% SDS, 0.1% NaDOC, 1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 20 mM HEPES), 1x wash buffer 2 (0.1% SDS, 0.1% NaDOC, 1% Triton X-100, 0.5 M NaCl, 1 mM EDTA, 20 mM HEPES), 1x wash buffer 3 (0.25 M LiCl, 0.5% NaDOC, 0.5% NP-40, 1 mM EDTA, 20 mM HEPES), and 2x wash buffer 4 (1 mM EDTA, 20 mM HEPES). Samples were treated with 10 µg RNase A in 100 µL for 20 min at 37°C. Elution was carried out in 2x200 µL elution buffer (0.1M NaHCO3, 1% SDS) for 2x15 min at room temperature with upside-down rotation. Samples were then treated with 60 µg Proteinase K for 1 hour at 55°C and subjected to decrosslinking in 300 mM NaCl by overnight incubation at 65°C. DNA was purified by standard PCI/chloroform extraction and ethanol precipitation and resuspended in 60 µL molecular biology-grade water. For input samples, 0.7% sonicated material was processed in parallel to the ChIP-seq samples from the RNase-treatment step.
Sequencing libraries were prepared according to the Illumina protocol, and sequenced on an Illumina Hiseq4000 (RBPJ, Notch1) or Illumina Nextseq500 (RelA, H3K27ac) machine at the Genomics Core Facility at Oslo University Hospital, Norway.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
 
Data processing ChIP-seq reads were aligned to the hg19 reference genome using bowtie2 version 2.2.6
Duplicate reads were removed using samtools rmdup
Peak calling was done using macs2 version 2.1.1.20160226
Genome_build: hg19
Supplementary_files_format_and_content: bigWig for viewing in genome browser; bed with for peaks (five columns: chromosome, start, end, peak name, peak summit position)
 
Submission date Oct 03, 2016
Last update date May 15, 2019
Contact name Guttorm Haraldsen
E-mail(s) guttorm.haraldsen@rr-research.no
Organization name Oslo University Hospital
Department Department for Pathology
Lab LIIPAT
Street address Sognsvannsveien 20
City OSLO
State/province OSLO
ZIP/Postal code 0027
Country Norway
 
Platform ID GPL18573
Series (2)
GSE85987 Inhibition of endothelial Notch signaling attenuates inflammation
GSE87552 Inhibition of endothelial Notch signaling attenuates inflammation [ChIP-Seq]
Relations
BioSample SAMN05858817
SRA SRX2207681

Supplementary file Size Download File type/resource
GSM2333689_Input_S1HA1h_nd.bw 103.3 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap