GSM5851371: Mettl7a1 sub polysomal fraction F; Mus musculus; RNA-Seq
One milliliter fraction was collected monitoring the absorbance at 260 nm using a Density Gradient Fractionation System by Teledyne ISCO with sensitivity set to 0.2. Using the proﬁle of the 260 nm absorbance, fractions corresponding to free ribosomal subunits (40S and 60S) and monosomes (80S, considered as not translating), separately from fractions corresponding to light polysomes (2–5 ribosomes) and heavy polysomes (>6 ribosomes) were pooled together for RNA extraction (see below). Proteins were isolated from each fraction through TCA/acetone precipitation. The same volume of 100% ice-cold acetone and 1/10th of TCA were added to each fraction. The samples were placed at -80°C overnight and then centrifuged at 16000 g for 10 min at 4°C. Pellets were washed three times with 1 ml of 100% ice-cold acetone, dried at RT for 5 min and resuspended in SDS-PAGE loading buffer. Illumina Stranded Total RNA Prep with Ribo-Zero Plus