GSM6784497: shPRRC2B polysome bio rep 3; Homo sapiens; OTHER
For total RNA extraction, cells were lysed by 1000 ml of Trizol (Qiagen), then mixed with 200 ml of chloroform.  The mixture was spun down at 16,000 g for 10 min. RNA was precipitated from the aqueous layer by adding two volumes of isopropanol and spinning down at 16,000 g for 10 min. The pellet was washed twice with 70% ethanol, left to dry, and resuspended in nuclease-free water. For ploysome-associated RNA extraction, 400 ml of each fraction was mixed with an equal amount of chloroform: phenol: isoamyl alcohol and 0.1 x of 3M sodium acetate (pH 5.2), then spun down at 16,000 g for 10 min. The upper aqueous layer was mixed with 3 volumes of 100% ethanol and 0.1 volume of 3M sodium acetate (pH 5.2) and left to incubate at -20°C overnight. The solution was then spun at maximum speed for 30 min to pellet the RNA, which was washed twice with 70% ethanol and resuspended in nuclease-free water. RNA libraries for RNA-seq were prepared using NGS Library Prep Kit following manufacturer's protocols.