GSM8635066: Control_S7; Homo sapiens; OTHER
After the enzymatic removal of proteins, total nucleic acids were isolated, and DNA and RNA were fractionated using the standard TRIzolTM protocol (Rio et al., 2010). The nucleic acid fractions from the aqueous phase (RNA) and chloroform-water interphase were ethanol-precipitated separately. These fractions were then re-fractionated through Zymo- SpinTM columns (ZYMO-RESEARCH CORP, Irvine, CA, USA) and eluted or further purified via chloroform extraction. Ethanol precipitated interface materials underwent overnight incubation at 56°C in a buffer containing 20 mM Tris (pH 8), 50 mM EDTA, 0.5% SDS, 20 µM dithiothreitol, and 400 µg/mL Proteinase K. Free RNA was separated, and DNA/RNA hybrids were further purified following DNase digestion and additional purification using Zymo-SpinTM columns (ZYMO-RESEARCH CORP, Irvine, CA, USA). Extracts were treated with RNase (to remove all RNAs), RNaseH (to remove only DNA-RNA hybrids), and DNase (to remove all DNA fragments). Following the enzymatic removal of proteins, total nucleic acids were fractionated into RNA and DNA-bound RNA. RNA was recovered  from blood samples after DNase digestion with amounts from 10 to 100 ng. High-throughput sequencing was performed on the Illumina HiSeq 2500 or MiSeq platform (Eurofins Medigenomix GmbH, Ebersberg, Germany) (Rassoulzadegan et al., 2021). RNA libraries were prepared from biological replicates of DNA-bound RNA fractions from the autistic patients.