title: GSM7848077: WCM399_Progression_SP55-59_cfDNA; Homo sapiens; Bisulfite-Seq
library_construction_protocol: Plasma was separated from whole blood with a standard double spin protocol and stored at -80°C. The input plasma volume ranged between 1mL and 2mL depending on the cohort.  For cfDNA extraction of samples processed in Trento, plasma was thawed at RT and immediately processed with the QIAamp Circulating Nucleic Acid Kit (QIAGEN) according to the manufacturer's protocol. The extracted cfDNA was eluted in 30 µL Tris HCl 10mM pH8 and quantified with Qubit dsDNA High Sensitivity Assay (Invitrogen); the quality was assessed using the Bioanalyzer High Sensitivity DNA Kit (Agilent). For samples processed at Weill Cornell Medicine (WCM, DFCI, and Alisertib trial) cfDNA was extracted from plasma using the NeoGeneStar Cell-Free DNA Purification kit per manufacturer's instructions. Briefly, for 2ml plasma samples, cfDNA was isolated via proteolytic digestion with 1μg of RNA carrier at 55°C for 30 minutes. cfDNA capture on the superparamagnetic particles was accomplished via the addition of 3 volumes (6mL) of LYS buffer, 0.8mL isopropanol, and 30μL NGSTM Beads. The capture of cfDNA was carried out via 30-minute room temperature incubation, 2x wash and 2x 80% EtOH, air drying, and elution in 30μL 10mM Tris, 0.1mM EDTA (pH 8.5). For PDX samples, 20-25 5µm slides of fresh-frozen tissue were cut using the HM525 NX cryostat (Thermo Scientific) and used for subsequent gDNA extraction. gDNA from PDX was extracted using the DNeasy Blood & Tissue Kit (QIAGEN), and gDNA from cell lines was extracted using the NucleoSpin Tissue Kit (Macherey-Nagel) according to the manufacturer's instructions and eluted in 100 µL Tris HCl 10mM pH 8. The gDNA was quantified using the Qubit dsDNA High Sensitivity Assay (Invitrogen). gDNA for libraries preparation was fragmented to a target size of 350 bp using the Covaris focused ultrasonicator M220. Based on the manufacturer's instructions, bisulfite conversion was performed on 5-25 ng of cfDNA and 200 ng of fragmented gDNA using the EZ DNA Methylation-Lightning Kit (Zymo). gDNA was re-quantified after bisulfite conversion using Qubit ssDNA High Sensitivity Assay (Invitrogen) and 100 ng of converted gDNA was used as input, whereas the whole converted cfDNA was used for libraries preparation for target sequencing with xGen Methyl-Seq DNA Library Prep Kit (IDT), following the kit's protocol with few modifications. All indexing PCR reagents were substituted with the HiFi HotStart Uracil+ ReadyMix (KAPA), and 11-12 PCR cycles were performed to achieve library yields higher than 500 ng for downstream experiments. For probes hybridization, 500 ng of each library of up to 12 samples were pooled together and captured using the xGen Hybridization and Wash Kit (IDT) following the xGen hybridization capture of DNA libraries protocol (version 4). The multiplexed libraries were dried together with the custom xGen Blocking Oligos and Human Cot DNA. Probes hybridization occurred during overnight incubation with custom xGen Lockdown Probes at 65°C. The captured DNA was amplified for 11 PCR cycles according to the panel size. Pre- and post-capture libraries were quantified using Qubit dsDNA High Sensitivity Assay (Invitrogen), and the quality was assessed with the Bioanalyzer High Sensitivity DNA Kit (Agilent). Libraries were sequenced on Illumina NovaSeq 6000 or MiSeq sequencing machines, with variable nominal depth based on the run and sample type (~100X for genomic DNA, ~1000X for cfDNA samples). Targeted Bisulfite-Seq