3T3L1-RNASeq-ShEmpty_D0_2
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 1uM 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. 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. For the Tet-On Gateway-compatible expression vector, the original vector was used as a negative control. For the KD, the lentiviral mammalian vector pLKO.1 containing specific shRNAs (three shRNAs per target were used, listed in the manuscript methods) along with the control shRNA (empty pLKO.1 plasmid) were obtained from Sigma. Finally, the ShRNA2 ZEB1 was not used in the ShRNA pool of ZEB1 as the robustness of the cells after treatment was low. Total RNA was isolated using a Qiagen RNAeasy plus mini kit according to the manufacturer's protocol by using either the RLT buffer lysis system or, for the RNA-seq samples, the TRIzol/Chlorophorm extraction procedure (Sigma). The RNA concentration and quality was determined using a nanodrop (1.8 _ A260/A280 _ 2.2) and by visual inspection of separated bands on agarose gels. 2 ug of total RNA was used for single strand cDNA synthesis using the SuperScript VILO cDNA Synthesis Kit (Invitrogen). After isolation of RNA, as described in the section “RNA isolation and quantitative PCR”, the Illumina Truseq RNA Sample Preparation kit v2 protocol (Illumina, USA) was followed using 500ng of RNA per sample as starting material. Half of the ligated reaction volumes were used for PCR (14 cycles) and the left one were kept at -80degreesC as a backup. Libraries were checked for quality and quantified using the Bioanalyzer 2100 (Agilent, USA), before being sequenced in barcoded pools of 16 samples on the Illumina Hiseq 2500 instrument (100 base paired-end sequencing, 4 lanes; Genomics sequencing facility, Nestle, Lausanne)).
ENA-FIRST-PUBLIC
2014-09-17T17:04:06Z
ENA-LAST-UPDATE
2018-03-08T17:36:38Z
External Id
SAMEA2517350
INSDC center name
EPFL
INSDC first public
2014-09-17T17:04:06Z
INSDC last update
2018-03-08T17:36:38Z
INSDC status
public
Submitter Id
E-MTAB-2538:3T3L1-RNASeq-ShEmpty_D0_2
broker name
ArrayExpress
cell line
3T3-L1
cell type
pre-adipocyte
common name
house mouse
developmental stage
pre-adipocyte
genotype
shEmpty
sample name
E-MTAB-2538:3T3L1-RNASeq-ShEmpty_D0_2
scientific_name
Mus musculus
specimen with known storage state
frozen specimen