GSM9574516: WT2 Hypothalamic neurons, INPUT (RT Dgkk specific); Mus musculus; OTHER
RNA was extracted from WT or Fmr1-KO neuron inputs (Input WT) or from CLIP samples (CLIP WT and CLIP Fmr1-KO) using phenol/chloroform (vol/vol) followed by chloroform/isoamyl alcohol (24:1) extraction and ethanol precipitation in the presence of 0.3 M sodium acetate. Total RNA was treated with DNase (Invitrogen, AM1907) and 500 ng of RNA was ribodepleted using the Mouse-Rat RiboPOOL kit (siTOOLs Biotech, catalog no. dp-K012-055) according to the manufacturer's protocol, followed by reverse transcription using SuperScript IV (ThermoFisher, 18090200). For the HITS-CLIP strategy in hypothalamic neurons, reverse transcription was conducted with random hexamers following the manufacturer's protocol (ThermoFisher, N8080127). For the iCLIP strategy in cortical neurons, a set of 18 Dgkκ-specific primers was utilized to cover the entire transcript of Dgkκ and optimize RNA-seq coverage. To enhance primer hybridization to Dgkκ mRNA, we modified the reverse transcription protocol: after 5 minutes of RNA denaturation at 65°C, a hot start at 55°C was initiated by adding the reverse transcriptase mix directly to the RNA. Unlike random hexamers, which can initiate reverse transcription at multiple sites, this method allows reverse transcriptase to start only at the specific positions defined by the primers. This approach helps in blocking reinitiation when encountering FMRP polypeptide generated by proteinase K digestion, resulting in an enrichment of transcript stop at the same 5' location. Generated cDNA was treated with 1 µL of RNase A (5 µg/mL) for 20 minutes at 37°C, then purified using RNA XP beads (Beckman Coulter) at a 1.8x ratio. The purified cDNA was sonicated in Covaris microtubes (AFA fiber Snap-cap). Finally, the fragmented cDNA was repurified with RNA XP beads and processed for MeDIP library preparation.