|
| Status |
Public on Sep 08, 2021 |
| Title |
CRE_ZT22_r3 |
| Sample type |
SRA |
| |
|
| Source name |
Liver
|
| Organism |
Mus musculus |
| Characteristics |
strain: Nr3c1fl/fl
aav injection: AAV-tbg-CRE
treatment: fed
circadian timepoint: ZT22
tissue/cell type: Liver
molecule subtype: rRNA depleted RNA
|
| Treatment protocol |
Nr3c1fl/fl mice were injected with AAV-tbg-GFP or AAV-tbg-CRE ten days before sacrifice. The primary hepatocytes were serum depleted in Gibo Medium 199 + GlutaMAX supplemented with 1 % P/S and 0.1 % HAS for 4 hours before cells were stimulated with 500 nM dex for 2.5 hours, human insulin for 2 hours or in a combination and RNA was harvested.
|
| Growth protocol |
Mice were housed at 12 hour light/dark cycles and trained to night restricted feeding. Primary hepatocytes were isolated from Male Sprague Dawley rats and seeded on collagen coated plates in Gibo Medium 199 + GlutaMAX (Gibco life technologies, cat: 41150-020) supplemented with 100 nM. Decadron (dex), 1 % P/S, 4 % FCS and 1 nM human insulin. 2-3 hours after isolation, the medium was changed to Gibo Medium 199 + GlutaMAX supplemented with 1 % P/S, 0.1 % FCS, 1 nM human Insulin and kept overnight.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA_seq: Primary hepatocytes: Cells were lysed in TRIzol-RNA lysis reagent (ThermoFisher). Liver tissue: Approximately 5 mg of liver tissues were homogenized using Ultra-Thorax in TRIzol-RNA lysis reagent. RNA was purified using EconoSpin columns (Epoc Life) according to manufacturer’s instructions.
ChIP-seq: Chromatin was prepared from 100-150mg formaldehyde crosslinked liver tissu. Crosslinked sonicated chromatin was IP'ed antibody and protein A/G agarose beads. DNA was extracted by phenol/choloroform.
Libraries from ChIP'ed DNA were constructed using the NEBNext Library prep kit according to manufacturer´s (NEB) instructions. Total RNA (1000 ng) was prepared for sequencing using ribosome depletion followed by random primed cDNA synthesis or polydT-mediated cDNA synthesis according to the manufacturer´s (Illumina) instructions. Subsequent library preparation was performed using the NEBNext RNA library preparation kit for Illumina.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
8064_S115
Sample_8064
processed data file: RNAseq_RAW_CT_GFP_CRE.txt
|
| Data processing |
RNA-seq and ChIP-seq sequencing data was aligned to mm10 or rn5 using STAR
RNAseq data was quantified by HOMER and normalised using DESeq2
GR ChIPseq peaks and bedgraph files were generated using HOMER
Genome_build: mm10 or rn5
Supplementary_files_format_and_content: Bedgraphs, peak bed file and text files with DESeq2 normalised RNAseq data
|
| |
|
| Submission date |
May 03, 2021 |
| Last update date |
Sep 08, 2021 |
| Contact name |
Lars Grøntved |
| E-mail(s) |
larsgr@bmb.sdu.dk
|
| Phone |
+45 24 60 14 06
|
| Organization name |
University of Southern Denmark
|
| Department |
Biochemistry and Molecular Biology
|
| Street address |
Campusvej 55
|
| City |
Odense |
| ZIP/Postal code |
5230 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL24247 |
| Series (1) |
| GSE173723 |
Impaired GR expression in liver disrupts feeding induced gene expression, glucose uptake and glycogen storage |
|
| Relations |
| BioSample |
SAMN18977426 |
| SRA |
SRX10754451 |
