title: Transcription profiling by high throughput sequencing of in vitro differentiated megakaryocytes
design: Transcription profiling by high throughput sequencing of in vitro differentiated megakaryocytes
library_construction_protocol: MKs were obtained by culture of CD34+ hematopoietic progenitor cells. In short, cord blood was collected with informed consent and CD34+ cells were prepared by magnetic bead selection. Purified cells were cultured (1x105 cells/ml) for 10 days in serum-free medium supplemented with human thrombopoietin and interleukin-1beta to differentiate into MKs.By day 10 more than 90% of cells stained positive for CD41 positive and negative for CD34. Total RNA was obtained by the Trizol method. Polyadenylated RNA was purified from this by two rounds of magnetic  Oligo dT25 selection (Dynabeads Oligo (dT)25, Invitrogen) in the presence of RNase inhibitor (New England Biolabs, Hitchin, UK), combined with DNase treatment (DNA-free, Ambion, Invitrogen). The mRNA was fragmented using RNA fragmentation reagent (Ambion, Invitrogen) and precipitated with added glycogen (Ambion, Invitrogen), then cDNA produced by Superscript Double Stranded cDNA synthesis kit (Invitrogen), replacing oligo-dT primer with random primer. Thereafter, library preparation was as per mRNA-sequencing kit (Illumina, Little Chesterford, UK), starting with end repair.
attributes.tag: Experimental Factor: CELL_TYPE
attributes.value: megakaryocyte