GX_MNX1_144h_rep2
Mus musculus
Protocols: Mouse embryonic stem cells (mES) were detached from gelatinesed flasks by addition of trypsin. Cells were disassembled from 3-dimensional gastruloids (initiated from mES cells and biased towards hemato-endothelial cell production) at 144 hours and 216 hours using the dissociation agent TrypLE Express. Cells suspensions were quenched with medium and centrifuged at 400 x g for 5 minutes to pellet for subsequent extraction. For colony forming assays, disassembled gastruloids cells from 144 hours or 216 hours were plated on Mouse Methylcellulose Complete Media (R&D Systems). Cells were first suspended in Iscove's Modified Dulbecco's Medium (IMDM, Gibco) with 20% FBS (Gibco) before addition to the methylcellulose medium. Cells were plated in duplicate 35 mm dishes with 2×10^5 cells/plate. Plates were incubated at 37°C and 5% CO2 for 10 days, when colonies were scored. Total RNA was extracted using Trizol (Invitrogen) and purified using Direct-zol RNA Purification Kits (Zymo Research). Library construction was performed by Novogene UK (Cambridge, UK). A minimum of 400 ng (20 ng/μl) of high-quality total RNA (extracted using Trizol, Invitrogen, and purified using Direct-zol RNA Purification Kits, Zymo Research) was supplied for sequencing. Novogene UK constructed cDNA libraries by purification of messenger RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, PCR amplification, and purification.
phenotype
MNX1 overexpression
organism
Mus musculus
organism
Mus musculus
collection date
not collected
scientific_name
Mus musculus
common name
house mouse
organism part
embryoid body
developmental stage
embryo stage
genotype
wild type genotype
isolate
not applicable
growth condition
gastruloid culture
cell_line
embryonic cell line
cell_type
embryonic stem cell
geographic location (country and/or sea)
not collected