MECs (EasySep, STEMCELL Technologies) were isolated from WT and CPEB2 KO animals (with two animals pooled per duplicate). Pellets were washed twice with cold Hanks’ balanced salt solution, lysed with RIPA buffer [50 mM tris-HCl (pH 8), 150 mM NaCl, 1 mM MgCl2, 1% NP-40, 1 mM EDTA, 0.1% SDS, protease inhibitor cocktail, and ribonuclease inhibitors] and sonicated for 5 min at low intensity with Standard Bioruptor Diagenode. After centrifugation (10 min, 4°C), supernatants were collected, precleared, and immunoprecipitated (4 hours, 4°C) with 10 μg of anti-CPEB2 antibody (69) bound to 50 μl of Dynabeads Protein G (Invitrogen). Beads were washed and split for either protein or RNA extraction. For RNA isolation, beads were resuspended in 100 μl of proteinase K buffer with 70 μg of proteinase K (Roche) and incubated for 30 min at 42°C and 30 min at 65°C. RNA was extracted following standard phenol/chloroform protocol. Samples were processed at IRB Barcelona’s Functional Genomics Facility following standard procedures: Illumina Hi-Seq 2000 50–base pair single-end RIP-sequencing (RIP-seq) data for WT and CPEB2 KO in biological duplicates, as well as their respective input samples of MECs, were checked for general sequencing quality control and adapter contamination using the FastQC software version 0.11, and no relevant problems were found. Afterward, reads were aligned against the M. musculus University of California, Santa Cruz mm10 ribosomal RNA (rRNA) genome using Bowtie1 0.12.9 (70) with two mismatches and default options to identify and remove reads coming from potential rRNA contamination from downstream analysis. Curated (non-rRNA) reads were then aligned against the M. musculus mm10 reference genome using Bowtie2 2.2.2 (71), allowing for one mismatch and reporting the best alignment site per read. All samples reported >15 million aligned reads. Potential amplification artefacts (duplicated reads) were detected and removed with the sambamba software version 0.5.1 using default options. Binary tiled data file tracks for visual inspection in the Integrative Genomics Viewer (IGV) software were generated using igvtools version 2. Read counts at 3′UTR level (longest 3′UTR per gene, mm10 genome Ensembl, March 2017) were computed using the featureCounts function from the Rsubread package version 1.24.2 with options minMQS = 1. Then, an interaction analysis of WT and CPEB2 KO RIP samples and their respective input controls (RIPWT/InputWT versus RIPKO/InputKO) was performed with DESeq2 (72). Target 3′UTRs were selected using an interaction FC threshold of >1.5 and interaction Benjamini-Hochberg adjusted P < 0.1 (see table S1, high-confidence RIP target genes, n = 169). GO enrichment for selected targets was performed using the online Enrichr (73, 74) tool.

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