1 protocol
nucleic acid library construction protocol
CD150hi-HSC, CD150lo-HSC and CD150neg-MPP were sorted into 500 µL of TRIzol reagent (Life Technologies). RNA was isolated according to manufacturer’s instructions and precipitated with the addition of glycogen (New England BioLabs). The RNA was then treated with DNAse 1 (Ambion) and purified with the RNeasy MinElute Cleanup Kit (Qiagen). For RNA-Seq analysis, total RNA (10 ng) were converted into double-stranded cDNA using the Ovation® RNA Amplification System V2 (NuGen, CA) per manufacturer’s recommendations. The amplified cDNA products were then used to generate RNA-seq libraries using the TruSeq RNA Sample Preparation Kit v2 reagents (Illumina®, CA) per manufacturer’s instructions, with 10 PCR amplification cycles. Library quality and quantity were assessed by the Agilent DNA1000 Chip (Agilent, CA) and qPCR (Kappa Biosystems Inc, MA).