title: GSM5456053: Riboseq_invitro_Rptorflfl_org1; Mus musculus; OTHER
library_construction_protocol: Briefly, for in vitro analyses, organoids were generated from VillinCre.ERT2.RPL22.HA and VillinCre.ERT2Rptor.fl/fl.RPL22.HA animals. Around 120-150 plugs of 30 μl BME were used for each replicate. Cells were treated with 100 μg/ml cycloheximide for 3-5 min at 37°C and immediately incubated on ice for the remainder of the experiment. After collecting the cells, pellets were washed twice with cold PBS supplemented with 100µg/mL cycloheximide, resuspended in ice-cold lysis buffer (20mM Tris HCl pH 7.4, 10mM MgCl2, 150mM KCl, 1% NP-40, 100µg/mL cycloheximide and 1x EDTA-free proteinase inhibitor cocktail (Roche)) and incubated for 20 min on ice. Lysates were then centrifuged at max speed for 20 min at 4°C and the supernatants were collected. For in vivo analyses, Lgr5CreERT2RPL22.HA and Lgr5CreERT2Rptor fl/flRPL22.HA animals were euthanized by CO2 and small intestines were immediately dissected, flushed with cold PBS supplemented with 100 µg/mL of cycloheximide and snap frozen using liquid nitrogen. Frozen samples were then homogenized by pestle and mortar while submerged in liquid nitrogen and the resulting powder was resuspended in ice-cold lysis buffer and incubated on ice for 30 min. Finally, lysates were centrifuged at max speed for 20 min at 4°C and the supernatants were collected. For Riboseq, HA pulldown was performed followingly. All lysates were incubated with Pierce™ Control Agarose Matrix (ThermoFisher) for 20min at 4'C in order to remove any non-specific binding, following by an incubation with pre-washed 425 AntiHA.11 Epitope Tag Affinity Matrix (BioLegend) for 4 hours (in vitro samples) or overnight (in vivo samples) at 4'C. Tagged ribosomes were then eluted from the beads by incubating with 200µg/mL HA peptide (ThermoFisher Scientifc) for 15 min at 30'C with constant agitation. Non-protected RNA was digested with 10 ul of RNase I (ThermoFisher Scientific) for 40min at 25'C and the reaction was stopped by adding 13ul of SUPERASE (ThermoFisher Scientific). Ribosome protected fragments (RPFs) were finally purified using miRNeasy minikit (Qiagen) accoridng to manufacturer's instructions. Riboseq library preparation was performed as previously described (Loayza-Puch et al., 2013) with some modifications. To discard undigested RNA fragments, RPFs were run in a 10% TBE-Urea polyacrylamide gel and size selected between 19 nt and 32 nt using marker RNAs. 3' ends were dephosphorylated using T4 polynucleotide kinase (PNK) (NEB) and 1.5xMES buffer (150mM MES-NaOH, 15mM MgCl2, 15mM β-mercaptoethanol and 450mM NaCl, pH 5.5) and incubated at 37'C for 4h. After purifying RNAs with Trizol, 3'adpaters were ligated with T4 RNA ligase I (NEB) and incubated overnight at 24'C. After size-selecting the ligated products, 5' ends were phosporylated with T4 PNK and incubated at 37'C during 30min. 5' adapters were then ligated with T4 RNA ligase I and incubated 37'C for 2,5 hours. The RPFs containing both adapters were then size-selected one more time and rRNA depletion was performed by first denaturing the samples at 100'C for 1min followed by incubation with 10uM biotinylated oligos (5'-UGAUCUGAUAAAUGCACGCAUCCCCCC-3'; 5'-CGUGCGAUCGGCCCGAGGUUAUCUAGAGUCACCA-3'; 5'-AUUCCAUUAUUCCUAGCUGCGGUAUCCAGGCGGCUC-3'; 5'-GGGCCUCGAUCAGAAGGACUUGGGCCCCCCACGA-3'; 5'-UGGCUUCCUCGGCCCCGGGAUUCGGCGAAAGC-3'; 5'-ACGGACGCUUGGCGCCAGAAGCGAGAGCCCCUCGGG-3'; 5'-ACCCGGCUAUCCGGGGCCAACCGAGGCUCCUUCGGCG-3'; 5'-ACCGACGCUCAGACAGGCGUAGCCCCGGGAGGA-3'; 5'-GGCGGACGGGGGGAGAGGGAGAGCGC-3'; 5'-GGCGAGACGGGCCGGUGGUGCGCCCUCGGC-3'; 5'-CCAGAAGCAGGUCGUCUACGAAUGGUUUAG-3'; 5'-AUCCCCGAUCCCCAUCACGAAUGGGGUUCA-3'; for the in vivo samples also added the following: 5'-GCUCUGCUACGUACGAAACCCCGACC-3'; 5'-GUGUCGAGGGCUGACUUUCAAUAGAUCG-3'; 5'-AGAUCCAACUACGAGCUUUUUAACUG-3'; 5'-AACGCCACUUGUCCCUCUAAGAAGU-3'; 5'-CAAGUGCGUUCGAAGUGUCGAUGAUC-3') in 2xSSC buffer (ThermoFisher Scientific) for 15min at 37'C. Pre-washed MyOne Streptavidin C1 DynaBeads (ThermoFisher Scientific) were then incubated with samples in wash/bind buffer (2M NaCl, 1mM EDTA, 5mM Tris and 0.2% Triton X-100) for 30min at 37'C with agitation. Supernatants were collected and RPFs were purified and re-suspended in 8µL of RNase-free water. cDNA was synthesized with SuperScript III (ThermoFisher Scientific) according to manufacturer's instructions and using the RTP primer (seq). After purification with G50 columns (Merck), cDNA was amplified using Phusion High-Fidelity DNA Polymerase (ThermoFisher Scientific) for 18 cycles, with primers containing different indexes to allow for sequencing (see table). PCR products were purified using the QIAquick PCR purification kit (Qiagen) followed by a final size selection with a E-Gel SizeSelect II 2%, (ThermoFisher Scientific). All samples were accessed with the Agilent 2100 Bioanalyzer to assure high quality and quantify the molarity and the libraries were sequenced on the Illumina HiSeq2500.