title: NextSeq 500 paired end sequencing; Transcriptomic profiling of human neuromesodermal-like cells and spinal cord progenitors derived in vitro from H9 ES cell line
design: Transcriptomic profiling of human neuromesodermal-like cells and spinal cord progenitors derived in vitro from H9 ES cell line
library_name: sd3/nmp 4_p
library_construction_protocol: Human ES cells (H9, sa181, sa121) and human iPS cells (ChIPS4) were maintained as feeder-free cultures in DEF-based medium (Cellartis DEF-CS) supplemented with bFGF (30 ng/mL, Peprotech) and Noggin (10 ng/ml,Peprotech) on fibronectin coated plates, and enzymatically passaged using 13TryLE select (thermofisher). For passaging, the medium was supplemented by addition of the Rho kinase inhibitor Y-27632 (10 mM, Tocris). For differentiation assays, PSC were plated on Geltrex matrix (20μg/cm2,LifeTechnologies) at a density of 40000 cells/cm2 and shifted to N2B27medium after 24h. Cells are differentiated for 3 days in N2B27 supplemented by 3 μM Chiron99021 (Tocris) and 20 ng/ml bFgf (PeproTech) to obtain NMPs.For further differentiation, NMPs were passaged using PBS-EDTA 0.5 mM and seeded back at controlled density (200000 cells cm2) on Geltrex (20μgcm2,LifeTechnologies) in the presence of Y-27632 (10 mM, Tocris). From passaging, cells are then cultured in N2B27 containing 100 nM RA for the indicated time to obtain later stage progenitors. In addition to the above, 50 ng/mL Nogginand 10μM SB431542 are added from day 2 to day 4. Total RNA was extracted using the RNEasy mini kit (Qiagen), following themanufacturer's instructions, with the addition of a DNAse digestion stepperformed on the column for 15 min with RQ1-DNase (Promega). RNAconcentration was measured on a Qubit device using Qubit RNA BR assay kit(ThermoFisher), and quality was checked on a TapeStation instrument(Agilent). Individually labelled libraries were prepared from 1 μg of RNA per sample using the TruSeq® Stranded mRNA Library prep kit (Illumina), according to the manufacturer's instruction. 2 μL of Spike-ins (1/100 dilution ERCC Spike-in controls Mix1) was added per sample.
attributes.tag: Experimental Factor: cell type
attributes.value: neuromesodermal progenitor cell