title: GSM8010791: CCHMC2016-7 Patient BM; Homo sapiens; RNA-Seq
library_construction_protocol: The RNA was extracted using the mirVana miRNA Isolation Kit (Thermo Fisher, Grand Island, NY) with the total RNA extraction protocol. In brief, cell lysates stored at -80C in Lysis/Binding Buffer were sent to the University of Cincinnati Genomics, Epigenomics and Sequencing Core for total RNA extraction according to the protocol and eluted with 60 µl Elution Buffer. The RNA concentration was measured by Nanodrop (Thermo Scientific, Wilmington, DE) and its integrity was determined by Bioanalyzer RNA 6000 Pico Kit (Agilent, Santa Clara, CA). NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs, Ipswich, MA) was used for poly(A) RNA purification with 50 uL total RNA as input. SMARTer Apollo NGS library prep system (Takara, Mountain View, CA) was used for automated poly(A) RNA isolation. The library for RNA-seq was prepared by using NEBNext Ultra II Directional RNA Library Prep kit (New England BioLabs, Ipswich, MA). During the second cDNA synthesis dUTP was incorporated to maintain strand specificity. In short, the isolated poly(A) RNA or rRNA/globin depleted RNA was fragmented (~200 bp), reverse transcribed to the 1st strand cDNA, followed by 2nd strand cDNA synthesis labelled with dUTP. The cDNA was then end repaired and dA-tailed and ligated to the adapter. The dUTP-labelled 2nd strand cDNA was enzymatically removed to maintain strand specificity. After indexing via PCR (13 cycles) enrichment, the amplified libraries together with the negative control were cleaned up for QC analysis. To check the quality and yield of the purified library, one µL library was analyzed by Bioanalyzer (Agilent, Santa Clara, CA) using DNA high sensitivity chip. To accurately quantify the library concentration for the clustering, the library concentration was qPCR measured by NEBNext Library Quant Kit (New England BioLabs) using QuantStudio 5 Real-Time PCR Systems (Thermo Fisher, Waltham, MA). read length: 51