GSM9598170: Input_1; Mus musculus; RNA-Seq
For RIP-seq, cortices were dissected from P0 heterozygous Celf2 KI mice, dissociated, and subjected to RIP using the EZ-Magna RIP RNA Immunoprecipitation Kit (Millipore) according to the manufacturer's instructions with modifications. Briefly, the cytoplasmic fraction was prepared as described above and supplemented with protease and RNase inhibitors. An aliquot was reserved for RNA extraction as the input control. The remaining supernatant was precleared with protein A/G beads (Bio-rad) and incubated with 10 μg of rabbit anti-CELF2 antibody or normal rabbit IgG for 3 hours at 4°C. Beads were washed, and co-precipitated RNAs were isolated for RNA sequencing. Total RNA was isolated from three biological replicates each of input, IgG control, and Celf2 immunoprecipitates, extracted by phenol/chloroform, and subjected to rRNA depletion using the NEBNext rRNA Depletion Kit (NEB). Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit (NEB) and sequenced on a NextSeq 500 instrument (Illumina). Sequencing quality was assessed using FastQC (v0.11.9), and adapters and low-quality bases were trimmed using fastp (v0.23.4). Residual rRNA reads were identified and removed using Ribodetector (v0.3.1).