2024-06-01
ENA first public
ERC000011
ENA-CHECKLIST
SAMEA114578463
External Id
University of Bergen
INSDC center name
2023-11-03T20:08:36Z
INSDC last update
public
INSDC status
ERS16641272
SRA accession
E-MTAB-13524:Irx3_ChIP_seq_iWAT_Day_1_parallel_a
Submitter Id
6 to 10
week
age
ArrayExpress
broker name
primary cell line
cell_line
preadipocyte
cell_type
not collected
collection date
house mouse
common name
Protocols: Mouse inguinal and gonadal white adipose tissues where excised from 6-10 weeks old C57BL/6NJ (B6N) mice fed ad libitum with SDS maintenance chow (RM3, 3.6 kcal/g). Primary preadipocytes were isolated from the WAT depots by by collagenase digestion and centrifugation to separate floating adipocytes from the SVF pellet. The SVF was resuspended in DMEM GlutaMax culture medium supplemented with 10% FBS and 1% PEST. The cell suspension was filtered through a 40 um mesh and pooled cells from 6-12 mice were seeded on a 10 cm dish at a density of 100.000 cells/mL. Cells were grown to confluence, and induced to differentiate 2 days post confluency by addition of 0.5 mM IBMX, 1 uM Dex, 5 ug/mL human Insulin to the growth media. On days -1 and 1 of differentiation, cells were fixated by addition of 1/10 volume of freshly prepared formaldehyde solution (11% formaldehyde, 0.1 M NaCl, 1mM EDTA pH 8.0 and 50 mM HEPES pH 7.9) to the culture media. After 15 minutes, the fixation reaction was stopped by addition of 1/20 volume of 2.5 M glycine. The cells were thereafter kept on ice and washed twice with 0.5% Igepal in PBS. In the second wash, 1mM PMSF was added. Pellets were snap-frozen in liquid nitrogen and stored at -80oC until shipment on dry ice to Active Motif for ChIP-sequencing Nucleic acid extraction was performed by Active Motif by in-house protocols based on public protocols:Park, P.J., 2009. ChIP–seq: advantages and challenges of a maturing technology. Nature Reviews Genetics 10: 669–80.Pepke, S., Wold, B., Mortazavi, A., 2009. Computation for chip-seq and rna-seq studies. Nature Methods 6(11 Suppl): S22-32.Furey, T.S., 2012. ChIP–seq and beyond: new and improved methodologies to detect and characterize protein–DNA interactions. Nature Reviews Genetics 13(12): 840–52. Nucleic acid library construction was performed by Active Motif by in-house protocols based on public protocols:Park, P.J., 2009. ChIP–seq: advantages and challenges of a maturing technology. Nature Reviews Genetics 10: 669–80.Pepke, S., Wold, B., Mortazavi, A., 2009. Computation for chip-seq and rna-seq studies. Nature Methods 6(11 Suppl): S22-32.Furey, T.S., 2012. ChIP–seq and beyond: new and improved methodologies to detect and characterize protein–DNA interactions. Nature Reviews Genetics 13(12): 840–52.
description
Day 1 of adipogenic differentiation
developmental stage
wild type genotype
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
subcutaneous adipose tissue
organism part
a
replicate
Mus musculus
scientific_name
C57BL/6NJ (B6N)
strain
Irx3_ChIP_seq_iWAT_Day_1_parallel_a
title