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| Status |
Public on Aug 29, 2018 |
| Title |
HUVEC_Notch1kd_IL1beta_RelA |
| Sample type |
SRA |
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| Source name |
Human umbilical vein endothelial cells (HUVECs)
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Human umbilical vein endothelial cells (HUVECs)
passages: Passage 4
state/ treatment: Confluent, treated with Notch1 siRNA and stimulated with IL-1β
chip antibody: anti-NF-κB/RelA, Santa Cruz Biotechnology sc-372
chip antibody lot # and amount used pr. chip-seq: F0514 (37.5 µL)
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| 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.
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| 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.
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| 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.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| 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)
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| Submission date |
Oct 03, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Guttorm Haraldsen |
| E-mail(s) |
guttorm.haraldsen@rr-research.no
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| Organization name |
Oslo University Hospital
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| Department |
Department for Pathology
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| Lab |
LIIPAT
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| Street address |
Sognsvannsveien 20
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| City |
OSLO |
| State/province |
OSLO |
| ZIP/Postal code |
0027 |
| Country |
Norway |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE85987 |
Inhibition of endothelial Notch signaling attenuates inflammation |
| GSE87552 |
Inhibition of endothelial Notch signaling attenuates inflammation [ChIP-Seq] |
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| Relations |
| BioSample |
SAMN05858811 |
| SRA |
SRX2207673 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2333681_RelA_N1DSG1h_nd.bw |
294.1 Mb |
(ftp)(http) |
BW |
| GSM2333681_RelA_N1DSG1h_peaks.bed.gz |
1.3 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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