GSM1895171: Mouse ESCs bc07 (E3); Mus musculus; RNA-Seq
Cytoplasmic mRNAs were separated on 10-50% sucrose gradients and fractionated into 12 fractions. Fractions were pooled corresponding to non-translated, low translation and high translation fractions. The bottom of the gradient was collected as fraction 12. Total RNA was treated with Ribominus kit (Life Technologies) as per manufacturer's instructions. Ambion SOLiD kit was utilized with protocol 4452437 (Revision B) opting for initial fragmentation using RNase III. After size-selection of the cDNA on Agencourt SPRI beads (as described), barcoding primers were used to amplify the libraries in a 14-cycle PCR reaction. No product was seen if reverse transcriptase was omitted in the reverse transcription step. Amplified libraries were checked for yield and quality on an Agilent Bioanalyzer chip and processed for SOLiD sequencing by the Biomolecular Resource Facility, Australian National University.