GSM2070081: Fraction2 rep2; Mus musculus; RNA-Seq
The supernatant was layered onto a 10mL linear sucrose gradient (10%-50% [w/v], supplemented with 10 mM HEPES pH 7.4,150 mM KCl,10 mM MgCl2, 0.5 mM DTT,100μg/ml cycloheximide), and centrifuged in a SW41Ti rotor (Beckman) for 120 min at 160,000 g at 4°C. Fractions were manually collected according to the A254 peaks that indicate the number of ribosomes. 3mg total RNA collected from each fraction were reverse transcribed using random primer (N15-oligo) tailed with 3’ part of Illumina TruSeq Universal Adaptor sequence. 5’ complete single-stranded cDNAs were captured based on a protocol from Takahashi et al, 2012 with minor modification. 5'end-sequencing