RNA Extraction and Sequencing from Cell Lines and Tissues

RNA from cell lines was obtained by lysing 1-2 million cells using 1 mL of Trizol (Invitrogen) then mixed with 1/5th volume of chloroform and centrifuged at 200×g for 5 min. The aqueous phase was collected and processed using a RNeasy Mini column (Qiagen). cDNA was synthesized from 1 μg of extracted RNA using SuperScript® III Reverse Transcriptase (ThermoFisher Scientific) with oligo(dT), random hexamers, and RNase inhibitor. Mouse brain tissue samples were collected from the cortex, cerebellum and striatum of heterozygous Mbd5^+/GT mice and wild-type controls at 8 weeks. Tissues that were previously frozen at −80°C were thawed overnight at −20°C submerged in RNAlater®-ICE Frozen Tissue Transition Solution (ThermoFisher Scientific) enabling easy cutting and extraction of high-quality RNA. RNA from tissues was obtained by lysing in 1 mL of Trizol (Invitrogen) using metallic pellets (Qiagen) and a tissue lyser, then mixed with 1/5th volume of chloroform and centrifuged at 200×g for 5 min. The aqueous phase was collected and mixed with isopropanol and centrifuged to obtain a pellet that was then washed with 75% ethanol and air dried and resuspended in RNAse-free water. Each tissue type was collected on the same day to avoid batch effects. The RNAseq library was prepared as previously described [29 (link)].