title: GSM7212173: RiboSeq_L22_PD_SCd_3; Mus musculus; OTHER
library_construction_protocol: Briefly, intestinal organoids were generated from VillinCreERT2RPL22.HA mice, 150 plugs were used for each replicate. Translating ribosomes were stalled by treating cells with 100 μg/ml cycloheximide for 3–5 minutes at 37 °C and immediately incubating them on ice for the remainder of the experiment. After collecting the cells, pellets were washed twice with cold PBS supplemented with 100 µg/mL cycloheximide, resuspended in ice-cold lysis buffer (20 mM Tris HCl pH 7.4, 10 mM MgCl2, 150 mM KCl, 1% NP-40, 100 µg/mL cycloheximide and 1x EDTA-free proteinase inhibitor cocktail (Roche)) and incubated for 20 minutes on ice, followed by mechanical disruption with a 25G syringe. Lysates were then centrifuged at max speed for 20 minutes at 4 °C and the supernatants were collected. Lysates were precleared with Pierce™ Control Agarose Matrix (Thermo Fisher Scientific) for 20 minutes at 4°C, and immunoprecipitated with pre-washed AntiHA.11 Epitope Tag Affinity Matrix (BioLegend) for 4 hours at 4oC. Beads were washed twice with ice-cold lysis buffer and twice with ice-cold wash buffer (20 mM Tris HCl pH 7.4, 10 mM MgCl2, 350 mM KCl, 1% NP-40, 100 µg/mL cycloheximide and 1x EDTA-free protease inhibitor cocktail (Roche)). Tagged ribosomes were then eluted by incubating the beads with 200 µg/mL HA peptide (Thermo Fisher Scientific) for 15 minutes at 30oC with constant agitation. Digestion of non-protected RNA was performed with 10 µl of RNase I (Thermo Fisher Scientific) for 40 minutes at 25 °C and the reaction was stopped by adding 13 µl of SUPERASE (Thermo Fisher Scientific). Lastly, ribosome-protected fragments (RPFs) were purified using miRNeasy minikit (Qiagen), following manufacturer's instructions. Ribosome Protected Fragments ranging from 19 to 32 nucleotides were size-selected using a 10% TBE-Urea polyacrylamide gel. 3' and 5' ends were modified accordingly and the respective adapters were ligated with a T4 RNA ligase I (New England Biolabs). The final products were size-selected one last time and rRNA depletion was performed using custom-made biotinylated oligos together with MyOne Streptavidin C1 DynaBeads (Thermo Fisher Scientific). Purified RPFs were then used to synthesize cDNA with SuperScript III (Thermo Fisher Scientific), according to the manufacturer's instructions, and using the RTP primer (Table yy). After purification with G50 columns (Merck), cDNA was amplified using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific), with primers containing different RP indexes to allow for sequencing. PCR products were purified with a QIAquick PCR purification kit (Qiagen) and size-selected one last time with an E-Gel SizeSelect II 2%, (Thermo Fisher Scientific).