3t3D0_Zeb1_1
Mus musculus
Protocols: 3T3-L1 mouse fibroblast cells obtained from ATCC, were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal calf serum (FCS, AMIMED), with 1X penicillin/streptomycin solution (Invitrogen) in a 5% CO2 humidified atmosphere at 37degreesC and maintained at less than 80% confluence before passaging. Differentiation of 3T3-L1 cells was induced by exposing two-day post-confluent cells (day 0) to DMEM containing 10% FCS supplemented with 1_uM dexamethasone, 0.5_mM 3-isobutyl-1-methylxanthine and 167 nM insulin (Sigma), a medium called MDI. After two days (day 2), cells were washed with Dulbecco's Phosphate-Buffered Saline 1x (PBS, Amimed) and were fed with DMEM containing 10% FCS and 167 nM insulin. Two days later (day 4), the media was changed to 10% FCS/DMEM. Full differentiation is usually achieved by day 6. ZFP277-HA overexpressing cells: For stable and selected lines, 3T3-L1 cells at a density of 5 x 104 cells were transduced with 10 ul viral particles in a 6-well plate. After 12 hours, the medium was changed and puromycin (Life Technologies, 2 _g/ml) was added after 72 hours to select stably transduced cells. If the cell confluence reached 80%, the cells were split and transferred into larger dishes. After every two days, puromycin selection media was changed and the stably transduced cells were selected for two weeks before performing actual experiments. 3T3-L1 stably selected cells were collected at days -2, 0, 2 and 4. The cells were fixed as described previously (Raghav et al., Mol Cell Biol 2012) and stored at -80�C. Ten million cells were used for each immunoprecipitation (IP). The ChIP experiment was performed as described previously in Raghav et al. (2012), under �Chromatin Immunoprecipitation of SMRT� in the Supplementary materials. A ZEB1 antibody (Santa Cruz, sc-25388, 10 _g per IP) and a rabbit isotype control IgG (Santa Cruz, SC-8994) was used for each time point. The DNA was stored at -20�C till verification of ChIP enrichment by qPCR and ChIP-seq library preparation. Multiplex libraries were prepared using barcoded adapters (see table below) for each sample following the protocol described in (Raghav and Deplancke, 2012) with slight modifications. In brief, ChIP-DNA fragments 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 the ligation incubation, the DNA was cleaned up using AMPure XP Beads (Agencourt) and eluted in 12 ul. These purified, ligated DNA fragments were separated on a 2% agarose gel to select 200-500 bp-sized DNA fragments and DNA was subsequently isolated from gel slices using a Qiagen gel extraction kit. The gel-extracted DNA was then amplified for 17 cycles by PCR using high fidelity Phusion hot start polymerase (NEB). The concentration and quality of purified, amplified DNA were estimated using respectively a Qubit dsDNA high sensitivity kit (Invitrogen) and a high sensitivity DNA assay Bioanalyzer 2100 (Agilent).
ENA-FIRST-PUBLIC
2014-09-17T17:04:06Z
ENA-LAST-UPDATE
2018-03-08T17:30:14Z
External Id
SAMEA2729988
INSDC center name
EPFL
INSDC first public
2014-09-17T17:04:06Z
INSDC last update
2018-03-08T17:30:14Z
INSDC status
public
Submitter Id
E-MTAB-2537:3t3D0_Zeb1_1
broker name
ArrayExpress
cell line
3T3-L1
cell type
pre-adipocyte
common name
house mouse
genotype
wild type
sample name
E-MTAB-2537:3t3D0_Zeb1_1
scientific_name
Mus musculus
specimen with known storage state
frozen specimen