NextSeq 550 sequencing; ChIP-seq based genome wide profiling of the histone mark H3K27ac and transcription factor Nuclear Factor 1 (NFI) in a differentiating murine brown preadipocyte cell line (IBA)
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
No description
Antigen
NA
Cell type
Cell type Class
Adipocyte
Cell type
Brown preadipocytes
NA
NA
Attributes by original data submitter
Sample
ENA first public
2017-09-21
ENA last update
2017-09-11
ENA-CHECKLIST
ERC000011
External Id
SAMEA104289171
INSDC center alias
SV-IBI-EPFL
INSDC center name
SV-IBI-EPFL
INSDC first public
2017-09-21T17:01:36Z
INSDC last update
2017-09-11T11:24:19Z
INSDC status
public
Submitter Id
E-MTAB-5159:IBA_Day0_NFI
broker name
ArrayExpress
cell line
immortalised brown preadipocyte
cell type
brown preadipocyte
common name
house mouse
progenitor cell type
brown preadipocyte
sample name
E-MTAB-5159:IBA_Day0_NFI
Sequenced DNA Library
library_name
IBA_Day0_NFI_s
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Murine derived brown pre-adipocytes (IBA) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) supplemented with 10% fetal calf serum (FCS, AMIMED), with 100x penicillin/streptomycin/glutamine (100x Pen/Strep/Glutamine, Life Technologies) in a 5% CO2 humidified atmosphere at 37C and maintained at less than 80% confluence before passaging Differentiation of IBAs was induced by exposing confluent cells (Day 0) to DMEM containing 10% FCS supplemented with 0.5 mM 3-isobutyl-1-methylxanthine, 1 uM dexamethasone, 1.96 nM insulin (Sigma, stock solution of 167uM prepared in PBS), 125 uM indomethacin (Sigma, stock solution of 18mM prepared in absolute ethanol), 1nM T3 (3,3’,5-Triiodo-L-thyronine sodium salt, Sigma, prepared in 45mM KOH). After exactly 28 hours, the induction medium was replaced with adipocyte differentiation medium consisting of 1nM T3 and 1.96 nM insulin Murine brown pre-adipocytes were collected at Day 0, 2 hours, Day 1, Day 2 and Day 4. The cells were fixed as described previously (Raghav and Deplancke, 2012) and stored at -80C. Ten million cells were used for each IP. The ChIP experiment was performed as using magnetic beads protocol (Dynabeads) (H3K27ac and NFI). Multiplexed libraries were prepared using barcoded adapters for each sample following the protocol described in (Raghav and Deplancke, 2012) with slight modifications. In brief, ChIP-DNA fragments (5-10ng) were end-repaired using an End-IT DNA end repair kit (Epicentre Technologies) followed by addition of an A-base and ligation of bar-coded adapters. After ligation incubation, DNA was cleaned up using AMPure XP beads (Agencourt). Two biological replicates for five time points (Day 0, 2h, Day 1, Day 2 and Day 4) were prepared for probing H3K27ac levels during differentiation, while one biological replicate at Day 0 and Day 4 was prepared for NFI.