GSM1319848: D11_mRNA-seq_replicate_1; Homo sapiens; RNA-Seq
Total RNA was purified with an ISOGEN (Nippon Gene) reagent from primary human erythroid cells after 4, 8, 11 or 14 days of ex vivo differentiation of CD34+ cells. The integrity and quality of RNA samples were assessed by the RNA integrity number (RIN) determined with an Agilent 2100 Bioanalyzer (Agilent Technologies). RINs of all the RNA samples used in this study were 10, indicating RNA of the highest integrity. The construction of the mRNA-seq libraries was performed according to standard instructions from Illumina. In brief, poly-A+ RNA was purified by poly-dT oligo-attached magnetic beads from 10 µg total RNA extracted from primary human erythroid cells after 4, 8, 11 or 14 days of ex vivo differentiation, and then sheared into short fragments of 200-300 bp in length by heating in the presence of divalent Zn2+ cations. These RNA fragments were reverse-transcribed using random primers into double-stranded cDNA fragments, which were end-repaired and then subjected to addition of a single adenine followed by ligation with an Illumina adapter to both ends. After PCR amplification, cDNA fragments approximately 200 bp in length were selected to generate a library to be sequenced in a paired-end 72-bp sequencing format using the Illumina Genome Analyzer IIx system in the DNA sequencing core facility of the University of Michigan. Two replicate RNA samples at each differentiation stage (day 4, 8, 11, and 14) were prepared from independent CD34+-cell cultures to generate two biological replicates of mRNA-seq libraries and data sets.