title: GSM4890360: 509 Spine-2; Homo sapiens; OTHER
library_construction_protocol: The metastatic lesions were dissected with ex vivo BLI imaging, and the uninvolved tissues were removed. The surgery tools were sterilized with a bead-sterilizer following by 70% isopropanol between different lesions. The collected tissues was snap-frozen in the liquid nitrogen until DNA extraction. The tissues were then rinsed by the gDNA lysis buffer from Quick-DNA Miniprep Plus Kit (Zymo Research, D4068) and homogenized with Precellys Lysing Kit (Bertin Instruments, MK28-R). After the homogenization, samples were lysed at 55℃ for 3 h, followed by 0.33 mg/mL RNaseA treatment at 37℃ for 15 min. Nucleic acid was further extracted following the manufacturer's instruction. The final DNA was assessed by NanoDrop 2000 (Thermo Scientific) and human/mouse DNA ratio was examined by q-PCR with primers targeting human and mouse GAPDH gene, respectively. Barcodes were amplified by two rounds of PCR. The first round of PCR was performed with 100 ng genomic DNA using Platinum Taq DNA Polymerase (Invitrogen) with Barcode-For and Barcode-Rev primers in 15 cycles. The second round of PCR were performed in a real-time setting and stopped in mid-exponential phase using PowerUp SYBR Green Master Mix (Thermo Fisher) with Barcode-P5-For and Barcode-P7-Rev primers. PCR products were then column-purified with QIAquick PCR purification Kit (QIAGEN) and assessed with Qubit. The NEBNext Multiplex oligos for Illumina (Dual index primer set 1,NEB, E7600S) and the NEB library preparation kit for Illumina (NEB, #E7645S) were used for library preparation as previously described (Kalhor et al., 2017).