title: Cord Blood derived eosinophil at day 24 CB106
design: Library Construction / Experimental Design     Total RNA was extracted using TRIzol® (Life technologies) according to the manufacturer protocol. For RNA-Seq, RNA libraries were created from each group using NEBNext® UltraTM Directinal RNA Library Preap kit for Illumina®. The first step in the workflow involved the removing of ribosomal RNA using RNAMiusTM Transcriptome Isolation kit (Lifetechnology). Following purification, the total RNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers and followed by second strand cDNA synthesis using DNA polymerase I and RNase H. The cDNA fragments were then processed through an end repair reaction by the addition of a single ‘A’ base, followed by ligation of the adapters. The products of these reactions were then purified and enriched with PCR to create the final cDNA library. The cDNA fragments were sequenced with HiSeq2500 (101 cycles PE lane) of Illumina Inc.