GSM9049717: Ribo Isogenic1; Homo sapiens; OTHER
Organoids were incubated in maturation medium supplemented with 10 μg/mL cycloheximide (CHX) for 5 min, washed twice with cold DPBS containing 20 μg/mL CHX, and snap-frozen in liquid nitrogen. Ten frozen organoids per sample were pooled and pulverized under liquid nitrogen using a mortar and pestle. The powder was collected in a 1.5 mL tube and resuspended in 400 μL Tissue Lysis Buffer (Immagina, #RL001-02) supplemented with 10% sodium deoxycholate (Thermo Scientific, #89904), 1 U/μl DNase I (Invitrogen, #AM2222), and 40 U/μl RiboLock RNase Inhibitor (Thermo Fisher Scientific, #EO0381), and centrifuged according to the manufacturer's protocol. The absorbance units (AU) of the samples were measured using a NanoDrop spectrophotometer at 260 nm. Ribosome-protected fragment pulldown. High Affinity ribosome beads (hiRB; Immagina, #GF001-04) were prepared for pulldown according to the functionalization protocol. Tissue lysates were subjected to nuclease digestion during the bead functionalization step. A fraction of each lysate was retained as an internal mRNA control (total mRNA). The digested lysates were incubated with functionalized beads for 70 min to allow ribosome binding. Ribosome-protected fragments (RPFs; length: 28–32 nt) were then purified using RNA Clean & Concentrator-5 (Zymo Research, #R1015). Library preparation. RPF library preparation was carried out according to the manufacturer's instructions and included the following steps: A. 5′ phosphorylation, B. Adapter ligation, C. Circularization, D. Reverse transcription, E. PCR amplification, F. PCR amplification 2, G. Library quality check, H. Sequencing. Sequencing was performed as a service by Immagina Biotechnology (Italy) on a NovaSeq 6000 instrument (Illumina).