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GEO help: Mouse over screen elements for information. |
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| Status |
Public on May 09, 2022 |
| Title |
rna_nt_rep3 |
| Sample type |
SRA |
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| Source name |
hepatocytes
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| Organism |
Mus musculus |
| Characteristics |
tissue: liver
strain: C57BL/6JOlaHsd
cell type: hepatocytes
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| Treatment protocol |
Cells were treated with glucagon (100nM) and corticosterone (1 µM) for 3h (RNA-seq) or 1h (ChIP-seq)
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| Growth protocol |
as described in PMID: 33111119
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA: Total RNA was isolated from primary mouse hepatocytes using NucleoSpin kit (Macherey-Nagel cat# 740955.25) according to the manufacturer’s protocol. ChIP: cells were cross-linked with 1% formaldehyde for 10 min at room temperature and quenched with 0.125M glycine. Crosslinked samples were washed in PBS, resuspended in ChIP lysis buffer (0.5% SDS, 10mM EDTA, 50mM Tris-HCl pH8) and sonicated (Bioruptor, Diagenode) to release 100-1000 bp fragments. Antibodies (4 μg per 300 μg chromatin) against H3K27ac (Active motif #39133) or GR (CST #3660) were conjugated to magnetic beads (Sera-Mag, Merck #GE17152104010150) for 2 h at 4°C. Chromatin was immunoprecipitated with antibody-bead conjugates over night at 4°C. Immunocomplexes were washed sequentially with the following buffers: low-salt buffer (0.01% SDS, 1% Triton x-100, 2 mM EDTA, 20mM Tris-HCl pH8, 150mM NaCl), high salt buffer (0.01% SDS, 1% Triton x-100, 2mM EDTA, 20mM Tris-HCl pH8, 500mM NaCl), low salt buffer and TE buffer (10mMTris-HCl, 1mM EDTA pH8). Chromatin was de-proteinized with proteinase K (Hy Labs EPR9016) for 2 h at 55°C and de-crosslinked over night at 65°C. DNA was subsequently phenol-chloroform purified and ethanol precipitated
For quality control of RNA yield and library synthesis products, the RNA ScreenTape and D1000 ScreenTape kits (both from Agilent Technologies), Qubit® RNA HS Assay kit, and Qubit® DNA HS Assay kit (both from Invitrogen) were used for each specific step. mRNA libraries were prepared from 1 µg RNA using the KAPA Stranded mRNA-Seq Kit, with mRNA Capture Beads (KAPA biosystems, cat# KK8421). ChIP DNA libraries were prepared using the KAPA HyperPrep Kit (KAPA biosystems, cat# KR0961).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Binary Base Call (BCL) output files from a Nextseq 500 machine were converted to FASTQ format, using BCL to FASTQ (bcl2fastq v2.20.0.422 Copyright (c) 2007-2017 Illumina, Inc.).
Fastq files were mapped to the mm10 mouse genome assembly using Bowtie2
Genome_build: mm10
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| Submission date |
Nov 21, 2021 |
| Last update date |
May 09, 2022 |
| Contact name |
Ido Goldstein |
| Organization name |
The Hebrew University of Jerusalem
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| Department |
Institute of Biochemistry, Food Science and Nutrition
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| Street address |
POB 12
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| City |
Rehovot |
| ZIP/Postal code |
7610001 |
| Country |
Israel |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE189271 |
The transcriptome, enhancer landscape and GR binding profile in primary mouse hepatocytes treated with glucagon and corticosterone |
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| Relations |
| BioSample |
SAMN23372115 |
| SRA |
SRX13183611 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5696891_rna_nt_rep3.bedGraph.gz |
47.1 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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