RNA sequencing of mouse embryonic stem cells (mES) and hemogenic gastruloids with MNX1 overexpression
RNA sequencing of mouse embryonic stem cells (mES) and hemogenic gastruloids with MNX1 overexpression
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.