title: Illumina NovaSeq 6000 paired end sequencing; RNA sequencing of mouse embryonic stem cells (mES) and hemogenic gastruloids with MNX1 overexpression
design: RNA sequencing of mouse embryonic stem cells (mES) and hemogenic gastruloids with MNX1 overexpression
library_name: mES_MNX1_rep1_p
library_construction_protocol: 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 endopoint (216 h) 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. 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/Î¼l) 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.