GSM4805192: embryo  KO 1; Mus musculus; RNA-Seq
Embryos were collected from females in synchronized pregnancies at 14.5 days post conception. Fetal livers were smashed by gentle pipetting in ice-cold PBS 1 x plus 2% FBS and cell suspension was filtered through a 70um cell strainer. Cells were counted and stained with Fc Block antibody followed by staining with FITC-conjugated anti-TER119 antibody. After wash and resuspension in an appropriate PBS volume, erythroid cells were sorted through BD FACS ARIA. Dead cells and debris were excluded by gating on forward scatter and side scatter morphologic parameters. RNA was prepared using QIAGEN RNeasy kit (#74106). Total RNA samples were processed according to DynabeadsTM mRNA DIRECTTM Purification Kit (Ambion) to isolate mRNA. The template library was prepared using the Ion Total RNA-Seq Kit v2 (Life Technologies). Quantity and size distribution of the library were analyzed using the Agilent Bioanalyzer 2100 DNA HS chip. Emulsion PCR using 10microl of 100pM library was performed using a OneTouch 2 instrument (Life Technologies) with an Ion PI Template OT2 200 kit following the manufacturer's instructions. The enrichment of template library was achieved using the Ion OneTouch ES enrichment system (Life Technologies). Ion Proton sequencer and IPv2 chip were prepared according to the manufacturer's recommendations.