title: GSM1833246: IC21; Homo sapiens; OTHER
library_construction_protocol: Bulk human peripheral blood plasma, containing contributions from an unknown number of healthy individuals, was obtained from STEMCELL Technologies (Vancouver, British Columbia, Canada) and stored in 2 ml aliquots at -80ºC until use. Individual human peripheral blood plasma from anonymous, healthy donors was obtained from Conversant Bio (Huntsville, Alabama, USA) and stored in 0.5 ml aliquots at -80ºC until use. Human peripheral blood plasma for 52 individuals with clinical diagnosis of Stage IV cancer was obtained from Conversant Bio or PlasmaLab International (Everett, Washington, USA) and stored in 0.5 ml or 1 ml aliquots at -80ºC until use. Human peripheral blood plasma for four individuals with clinical diagnosis of systemic lupus erythematosus was obtained from Conversant Bio and stored in 0.5 ml aliquots at -80ºC until use. Barcoded double-strand preparation sequencing libraries were constructed with the ThruPLEX-FD or ThruPLEX DNA-seq 48D kits (Rubicon Genomics), comprising a proprietary series of end-repair, ligation, and amplification reactions. Between 0.5 ng and 30.0 ng of cfDNA were used as input for all clinical sample libraries. Library amplification for all samples was monitored by real-time PCR to avoid over-amplification, and was typically terminated after 4-6 cycles. Single-strand ligation libraries were prepared from dephosphorylated cell-free DNA. For dephosphorylation, 2X CircLigase II buffer (Epicentre), 5 mM MnCl2, and 1U FastAP alkaline phosphatase (Thermo Fisher) and 0.5-10 ng fragments were incubated in a 20 ul reaction volume at 37ºC for 30 minutes. Fragments were then denatured by heating to 95ºC for 3 minutes, and were immediately transferred to an ice bath. The reaction was supplemented with the biotin-conjugated adapter oligo (5 pmol), 20% PEG-6000 (w/v), and 200U CircLigase II (Epicentre) for a total volume of 40 ul, and was incubated overnight with rotation at 60ºC, heated to 95ºC for 3 minutes, and placed in an ice bath. For each sample, 20 ul MyOne C1 beads (Life Technologies) were twice washed in bead binding buffer (BBB) (10 mM Tris-HCl [pH 8], 1M NaCl, 1 mM EDTA [pH 8], 0.05% Tween-20, and 0.5% SDS), and resuspended in 250 ul BBB. Adapter-ligated fragments were bound to the beads by rotating for 60 minutes at room temperature. Beads were collected on a magnetic rack and the supernatant was discarded. Beads were washed once with 500 ul wash buffer A (WBA) (10 mM Tris-HCl [pH 8], 1 mM EDTA [pH 8], 0.05% Tween-20, 100 mM NaCl, 0.5% SDS) and once with 500 ul wash buffer B (WBB) (10 mM Tris-HCl [pH 8], 1 mM EDTA [pH 8], 0.05% Tween-20, 100 mM NaCl). Beads were combined with 1X Isothermal Amplification Buffer (NEB), 2.5 uM oligo CL9, 250 uM (each) dNTPs, and 24U Bst 2.0 DNA Polymerase (NEB) in a reaction volume of 50 ul, incubated with gentle shaking by ramping temperature from 15ºC to 37ºC at 1ºC/minute, and held at 37ºC for 10 minutes. After collection on a magnetic rack, beads were washed once with 200 ul WBA, resuspended in 200 ul of stringency wash buffer (SWB) (0.1X SSC, 0.1% SDS), and incubated at 45ºC for 3 minutes. Beads were again collected and washed once with 200 ul WBB. Beads were then combined with 1X CutSmart Buffer (NEB), 0.025% Tween-20, 100 uM (each) dNTPs, and 5U T4 DNA Polymerase (NEB) and incubated with gentle shaking for 30 minutes at room temperature. Beads were washed once with each of WBA, SWB, and WBB as described above. Beads were then mixed with 1X CutSmart Buffer (NEB), 5% PEG-6000, 0.025% Tween-20, 2 uM double-stranded adapter 2, and 10U T4 DNA Ligase (NEB), and incubated with gentle shaking for 2 hours at room temperature. Beads were washed once with each of WBA, SWB, and WBB as described above, and resuspended in 25 ul TET buffer (10 mM Tris-HCl [pH 8], 1 mM EDTA [pH 8], 0.05% Tween-20). Second strands were eluted from beads by heating to 95ºC, collecting beads on a magnetic rack, and transferring the supernatant to a new tube. Library amplification for all samples was monitored by real-time PCR to avoid over-amplification, and required an average of 4 to 6 cycles per library.