Paxgene blood ccfdna tube

Peripheral blood (10 ml) was collected in PAXgene Blood ccfDNA tubes (Qiagen, Hilden, Germany). The collected whole blood was immediately centrifuged at 1900 × g for 15 min at room temperature (23°C to 27°C). The supernatant was collected and centrifuged again at 1900 × g for 10 min. The supernatant from the second centrifugation was considered the final plasma and was stored at −80°C until use. ctDNA was extracted from the plasma using a QIAamp Circulating Nucleic Acid Kit (Qiagen) according to the manufacturer's instructions.

Whole blood was collected in PAXgene Blood ccfDNA tubes (Qiagen, Cat. No. 768115) and centrifuged at 1900×g for 10 min at RT to isolate plasma. Plasma was centrifuged twice at 16,000×g for 10 min and stored at −80 °C until cfDNA extraction. Circulating cfDNA was extracted from 4 ml (ALS patients) or 8 ml (controls) of plasma using the QIAamp Circulating Nucleic Acid kit (Qiagen, Cat. No. 55114). Larger volumes of control blood were collected to ensure equal amounts of total cfDNA (compared to patients) were analyzed. cfDNA quality and concentration were assessed with an Agilent 2100 Bioanalyzer, using the Agilent High Sensitivity DNA kit (Agilent, Cat. No. 5067-4626). 10 ng of cfDNA were bisulfite-treated and purified using the EZ DNA Methylation-Direct Kit (Zymo Research Cat. No. D5020). Libraries for whole genome bisulfite-sequencing were generated using Accel-NGS^® Methyl-Seq DNA Library Kit (Swift Biosciences, Cat. No. 30024) and Accel-NGS Methyl-Seq Dual Indexing kit (Swift Biosciences, Cat. No. 38096), with eight cycles of indexing PCR. Libraries were quantified by qPCR with the Hyper Library Quantification kit (Kapa, Cat. No. KR0405) and paired-end sequenced on a NovaSeq 6000 System (Illumina).

Plasma samples were collected using PAXgene Blood ccfDNA tubes (#768165; Qiagen, Hilden, Germany). Blood samples were collected weekly during hospitalization following transplantation, and then every time the patients underwent medical examinations or transbronchial biopsies. A total of 372 plasma samples were obtained from 30 patients (average, 12,4/patient). Plasma was separated by centrifugation (2,000 ×g, 15 min, 18°C) and stored at −80°C in the Teseo Biobank of the Department of Medical Sciences of University of Turin (http://www.progettoeccellenzateseo.unito.it/) until further processing. Cell-free DNA (cfDNA) was extracted from 1 ml of plasma using MagMAX Cell free DNA isolation kit (#A29319, Applied Biosystems, Waltham, MA, United States) and stored at −20°C until analysis.