title: GSM5225574: SporeF2; Homo sapiens; OTHER
library_construction_protocol: 1-4 mL healthy human donor plasma (Vitalant) or 0.2-0.5 mL of mouse serum were thawed from -80ºC storage. Plasma or serum were spun at max speed (21000 rcf) at room temperature to pellet any cell debri. Supernatant was added to new tubes and cfDNA was extracted using the QIAGEN ccfMinElute kit (QIAGEN 55204). Eluates were eluted in 30 µL of nuclease-free water and directly added to the single-stranded DNA library protocol (SSP) or stored at -20ºC. The capture of cfDNA fragments from plasma or serum was performed as in Snyder et al. 2016 and Gansauge and Meyer 2013. In brief and with the following changes, cfDNA 1-10ng was dephosphorylated using FastAP (Thermo EF0651), denatured, and incubated overnight with CircLigaseII (Lucigen CL9025K) and 0.093-0.125 µM biotinylated CL78 at 60ºC with shaking every 5 min. Captured cfDNA fragments were denatured and then bound to magnetic streptavidin M-280 beads (Invitrogen 11205D)  for 30 min at room temperature with nutation. Beads were washed and second-strand synthesis was performed using Bst 2.0 DNA polymerase (NEB M0537) with an increasing temperature gradient 15-31ºC with shaking at 1750 rpm. Beads were washed and a 3' gap fill was performed using T4 DNA polymerase (Thermo EL0011) for 30 min at room temperature. Beads were washed and a double-stranded adapter was ligated using T4 DNA ligase (Thermo EP0062) for 2 hours at room temperature with shaking at 1750 rpm. Beads were washed and resuspended in 30 µL 10 mM TET buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 0.05% Tween-20). Beads were denatured at 95ºC for 3 min and cfDNA libraries were collected after immediate magnet separation. Quantitative real-time PCR was performed on cfDNA libraries using iTAQ Supermix (Bio-Rad 1725124) and Ct values were used to determine the number of PCR cycles needed to amplify each library. PCR was performed with KAPA HiFi DNA polymerase (Kapa Biosystems KK2502) using barcoded indexing primers adapted for Illumina sequencing on the Nova-seq. Primer dimers were removed from the libraries using 0.9X AMPure beads (Beckman Coulter A63881). Libraries were eluted in 0.1X TE and concentrations were determined using Qubit. The length distribution of each library was assessed by the Agilent Bioanalyzer using the D1000 or HSD1000 cassette. Libraries were then submitted for Illumina sequencing.