GSM9465581: CHLA-9, total RNA, replicate 3; Homo sapiens; RNA-Seq
Cells were cultured for 48h and harvested in cycloheximide-containing lysis buffer. Following mechanical lysis and flash-freezing, samples were fractionated along a 10-50% sucrose-gradient prepared with the BioComp Gradient Station (“LONG, Sucr, 10-50%, w/v” program) through ultracentrifugation (38.000 rpm, 2.30h, 4°C – TH-641 swinging bucket rotor). Samples were then scanned for the absorbance at 260 nm with the BioComp Gradient Station, and all the fractions were manually retrieved, flashed-frozen and subsequently stored at -80°C until use. For each sample, all the fractions corresponding to each polysome peak were pulled together, followed by RNA extraction and library preparation for RNA seq. Total RNA seq was isolated from the matched lysate that did not underwent fractionation. NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® was employed for RNA-seq library preparation.