title: Illumina HiSeq 2500 sequencing; Fractionation RNAseq in conjunction with Illumina TRUseq method
design: Fractionation RNAseq in conjunction with Illumina TRUseq method
library_construction_protocol: 3 µg of RNA was treated with TURBO DNase, followed by RNeasy Minelute RNA cleanup kit and RNA quaility was confirmed using Agilent 2100 Bioanalyzer with RNA Pico 6000 kit (RIN values >8). 0.5 µg of RNA was used for Lexogen QuantSeq-REV libraries. cDNA libraries were prepared according to Lexogen QuantSeq Rev method manufacturer's protocol, using a poly(T) primer for reverse transcription. The library was sequenced using Illumina HiSeq, producing 60 nt single-end reads and 10 nt index reads. Differentiation was induced by addition of BMP4 for 48hrs followin dissociation of colonies into single cells using accutase and subsequently seeding them on Matrige-coated 6-well plates containing mTESR1 medium  supplemented with 20% KnockOut Serum Replacement, Glutamax, nonessential amino acids, beta-mercaptoethanol, and 50 ng/ml BMP4. Medium was changed daily. RNA-seq
attributes.tag: Experimental Factor: fraction
attributes.value: nucleoplasm fraction