GSM9217012: cfDNA, ASD, non-obese, 6; Homo sapiens; Bisulfite-Seq
CfDNA was extracted from the supplied plasma aliquots with the QIAamp Circulating Nucleic Acids Kit (QIAGEN, 55114) by following recommended procedures and quantified with the Qubit DNA Assay Kit (ThermoFisher Scientific, Q32851). All purified cfDNA samples were also analyzed with the Bioanalyzer High Sensitivity DNA Kit (Agilent, 5067-4626) to verify sample concentrations and validate that samples contained only the small nucleic acids typical of cfDNA. Any samples containing nucleic acids of ≥200bp length were subjected to a size-selective clean-up with SPRIselect (Beckman Coulter, B23317) beads. CfDNA was bisulfite-converted using the EZ-DNA Methylation Direct Kit (Zymo Research, D5020) according to manufacturer recommendations. No fragmentation was performed, as cfDNA are naturally of a short length amenable to library preparation. Whole genome bisulfite sequencing libraries were then prepared from the bisulfite-converted cfDNA using the xGen Methylation-Sequencing DNA Library Preparation Kit (IDT, 10009860) with UDI Primer Pairs (IDT, 10005922) and an 11-cycle indexing PCR. Libraries were assessed for uniform size with Bioanalyzer High Sensitivity DNA Kit and pooled to 10nM concentration with a 10% PhiX spike-in. Pooled libraries were sequenced on 4 lanes with 150bp paired end reads of Illumina NovaSeq 6000 S4 (Illumina, 20012850) by the DNA Technologies Core at UC Davis.