title: GSM9324436: PolysomeProf, 42C, NonDepl, Fraction with Ribosome = 1; Mus musculus; RNA-Seq
library_name: GSM9324436
library_construction_protocol: For the sampling, cells were collected by scraping in PBS supplemented with cycloheximide (CHX) at 200μg/mL. Then the mixture was spun down at 500g for 3min in a centrifuge at 4°C. The supernatant was removed and the pellet was immediately subjected to lysis. The cell lysis and gradient preparation was performed as previously described in Rahaman, et. al. Cell Reports, 2025. For the lysis, cells were dissolved in 1X mammalian Polysome Lysis Buffer [20mM Tris-HCl, 10mM MgCl2, 1mM DTT, 50mM KCl, 200μg/mL cycloheximide, 2 cOmplete EDTA free Protease Inhibitor 100mL, 16U/mL RNasIN 1% v/v Triton X-100 ]. Lysis was carried out by passing the dissolved cells through a 23G syringe 10 times, followed by 10 cycles of bead-based lysis using Zirconia/Silica beads. The lysate was then clarified by a centrifugation for 5min at 10000g. For the ultracentrifugation, the A254 of the lysate was measured by mixing sample (or blank with Polysome Lysis Buffer) with water in a 1:20 volumetric ratio. 2–4 A254 unit equivalents of the sample was loaded onto the 10–50% sucrose gradient. Ultracentrifugation was carried out with a SW41Ti rotor (swing bucket) at 40000rpm (equivalent to 275,000g) for 2h at 4°C. The polysome profile was generated with BioComp Polysome Profiling machine. 25 fractions (450μL each) corresponding to different positions in the centrifuge tube were collected. For RNA purification, the fractions were pooled according to the peaks of A254 polysome profile (free fraction, 40S, 60S, monosome, etc.), and in vitro transcribed spike-in RNAs (prepared by in vitro translation using TranscriptAid T7 High Yield Transcription Kit) were added to each pool to serve as a volumetric control. Subsequently, the pools were subjected to phenol-chloroform extraction (Phenol: chloroform: isoamyl-alcohol = 25:24:1). Precipitation was carried out with 2.5 volumes of ethanol at −80°C overnight with 2μL of GlycoBlue as co-precipitant. RNA was reconstituted with 20μL of nuclease free water containing RNasIN at 1μL/mL (40U/mL). The purified RNA was then used for qRT-PCR or RNA-Seq. Stranded SMART-Seq (Takara)
identifiers.namespace: GEO
identifiers.id: GSM9324436_r1