NextSeq 500 paired end sequencing; RNA-seq analysis of substates of human pluripotent stem cells with mesoderm bias and early differentiating cells towards the mesoderm lineage
RNA-seq analysis of substates of human pluripotent stem cells with mesoderm bias and early differentiating cells towards the mesoderm lineage
10,000 cells from the given population are sorted into an eppendorf tube containing 800µL Trizol. Post sort, samples were sealed, vortexed and stored at -80°C until extraction. Total RNA was extracted from the aqueous phase of thawed Trizol (Thermo Fisher) lysates after addition of 160ul chloroform and precipitated by mixing an equal volume of isopropanol supplemented with 10ug of linear polyacrylamide (Sigma). The RNA pellet was washed twice with 70% ethanol and resuspended in water. Total RNA was quality controlled and quantified using Bioanalyzer RNA 6000 Pico chip kit (Agilent).  1.5ng total RNA was used with SMART-Seq.v4 kit (Clontech) to prepare cDNA libraries as per supplier instructions. cDNA libraries were purified with Agencourt AMPureXP (Beckmam) magnetic beads (at 0.8 bead to 1 cDNA ratio), washed twice with fresh 80% ethanol and eluted in EB buffer. Each of the cDNA libraries were quality checked using Fragment Analyzer  High Sensitivity Large Fragment kit (Agilent), and quantified using Qubit High Sensitivity DNA kit (Thermo Fisher). 150pg of each cDNA was used to prepare sequencing libraries with Nextera XT DNA Preparation Kit and Indexing primers (Illumina), but following a slightly modified version of the manufacturer's protocol involving 4-fold volume miniaturisation throughout (12.5ul final volume). Nextera-XT indexed sequencing libraries were purified with Agencourt AMPureXP magnetic beads (at 0.8 bead to 1 cDNA ratio), washed twice with fresh 80% ethanol and eluted in EB buffer. Each of the indexed libraries were quality checked using Fragment Analyzer High Sensitivity Large Fragment kit, and quantified using Qubit High Sensitivity DNA kit. Indexed sequencing libraries were pooled to be equimolar.
Experimental Factor: growth condition
Reverted in presence of LPA after 9 passages in PRIMO Plus
Experimental Factor: stimulus
none
Experimental Factor: sorted fraction
MIXL1-GFP(-)/SSEA-3(+)