AA/F2 - 3D
Mus musculus
Protocols: ESCs and EpiSCs were cultured according to standard conditions. Feeder-free ESCs were seeded on fibronectin (3-5 ng/ml)-coated plates in ESC medium containing LIF, PD0325901 (1 μM) and CHIR99021 (3 μM) (Axon Medchem, Axon 1386) (2i medium). The cells were re-fed the next day with 2i medium again (N2B27 medium (Yao et al., 2006) with LIF, PD0325901 and CHIR99021) to bring them to a naive state of pluripotency. The following day, the medium was replaced with EpiSC medium (N2B27 medium + Activin A (20 ng/ml) + FGF2 (10 ng/ml)). The medium was replaced with fresh EpiSC medium and fresh ligands every day, for 3 days (72 hours) to convert them to an EpiSC-state. Then, for PSM induction, the cells were fed with N2B27 medium containing CHIR99021 (3μM) either alone or along with FGF ligands, FGF2 or FGF8 (Low concentration: 10 ng/ml or High concentration: 250ng/ml). Medium was replaced along with fresh ligands every day for 2-3 days. In vitro: Cells were rinsed with PBS and trypsinised with cold Trypsin, spun-down at high speed (10,000 RPM) for 0.5 minute and re-suspended in cold Knock-out DMEM and placed on ice. In vivo: The embryos (E8,75) generated via tetraploid complementation from Tbx6-transgenic ESCs were dissected from uteri in PBS and transferred to M2 medium (Sigma) for staging and micro-dissection. The caudal ends (up to the presomite-2 region), containing the PSMs were dissected-out from embryos and pooled. Without much time-delay, the cells were sorted on FACS Aria II (Becton Dickinson) directly into RLT buffer containing β-mercaptoethanol, followed by total RNA isolation employing the RNeasy Micro Kit (Qiagen) and further processing. The unsorted cells were directly lysed with the RLT buffer in the cell-culture wells, after rinsing once with PBS. As a precautionary measure, to avoid genomic DNA, an extra step of DNase I (Roche) treatment was carried out. RNA was quantified using the spectrophotometer from either NanoDrop Technologies (unsorted cells) or Qubit (Invitrogen) (FACS-sorted cells). 'RNA-seq was performed using 80 ng of total RNA. The total RNA was quantified with Qubit fluorometer using the RNA HS kit (Invitrogen) and the quality was assessed with RNA Pico kit (Agilent Technologies) using an Agilent Bioanalzyer. Only samples with high RIN (RNA integrity number) scores (>9) and intact 28S-18S rRNA bands were processed. RNA-Seq library was prepared from total RNA using the ScriptSeq Complete Kit (Human/Mouse/Rat)-Low Input (SCL24H) (Illumina) according to manufacturer''s instructions. After library preparation and before sequencing, the libraries were quantified using the Qubit DNA HS kit (Invitrogen) and checked again on bioanalyzer using the DNA HS chips (Agilent Technologies). During library preparation, the rRNA-depleted sample-purification was done using the RNeasy MinElute Cleanup Kit (Cat. No. 74204, Qiagen) and cDNA-purification was done using MinElute PCR Purification Kit (Qiagen). For amplification, 15 cycles were used (using Failsafe enzyme). The quality of the cDNA libraries for sequencing was assessed using Bioanalyzer (Agilent Technologies). '
ENA-FIRST-PUBLIC
2016-06-01T17:01:03Z
ENA-LAST-UPDATE
2018-03-09T02:21:33Z
External Id
SAMEA3493437
INSDC center name
Department of Developmental Genetics, Max Planck Institute for Molecular Genetics
INSDC first public
2016-06-01T17:01:03Z
INSDC last update
2018-03-09T02:21:33Z
INSDC status
public
Submitter Id
E-MTAB-3784:AA/F2 - 3D
broker name
ArrayExpress
cell type
mouse embryonic stem cell
common name
house mouse
fraction
whole cell lysate
genetic modification
transgenic
genotype
Tbx6 reporter
growth condition
AA (Activin A) (20 ng/ml) and F2 (FGF2) (10 ng/ml) for 3 days
sample name
E-MTAB-3784:AA/F2 - 3D
scientific_name
Mus musculus
strain
C57BL/6/N