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Status |
Public on Jul 13, 2017 |
Title |
Lung cancer 13 |
Sample type |
SRA |
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Source name |
Lung cancer 13
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Organism |
Homo sapiens |
Characteristics |
sample name: lung514 tissue: blood target molecule: cell-free DNA
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Treatment protocol |
not applicable
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Growth protocol |
not applicable
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Extracted molecule |
other |
Extraction protocol |
Samples for healthy subjects were obtained from Stanford blood center. HCC and BrCa patients were recruited in a Stanford University Institutional Review Board-approved protocol. LuCa, PaCa, GBM, GaCa and CoCa patients were recruited in a Sichuan University Institutional Review Board-approved protocol. Blood was collected into EDTA-coated Vacutainers. Plasma was collected from the blood samples after centrifugation at 1,600 × g for 10 min at 4 °C and 16,000 × g at 10 min at 4 °C. cfDNA was extracted using the Circulating Nucleic Acid Kit (Qiagen). Whole blood genomic DNA was extracted using the DNA Mini Kit (Qiagen) and fragmented using dsDNA Fragmentase (NEB) into average 300 bp. DNA was quantified by Qubit Fluorometer (Life Technologies). Cell-free RNA was extracted using the Plasma/Serum Circulating and Exosomal RNA Purification Kit (Norgen). The extracted cell-free RNA was further digested using Baseline-ZERO DNases (Epicentre) and depleted using Ribo-Zero rRNA Removal Kit (Epicentre) according to a protocol from Clontech. cfDNA (10 ng) or fragmented genomic DNA (1 µg) spiked with amplicons (0.001 pg of each amplicon per 10 ng DNA) was end repaired, 3’-adenylated and ligated to DNA Barcodes (Bioo Scientific) using KAPA Hyper Prep Kit (Kapa Biosystems) according to the manufacturer’s instructions. Ligated DNA was incubated in a 25 µl solution containing 50 mM HEPES buffer (pH 8), 25 mM MgCl2, 100 µM UDP-6-N3-Glc (Active Motif), and 12.5 U βGT (Thermo) for 2 hr at 37 °C. After that, 2.5 μL DBCO-PEG4-biotin (Click Chemistry Tools, 20 mM stock in DMSO) was directly added to the reaction mixture and incubated for 2 hr at 37 °C. Next, 10 µg sheared salmon sperm DNA (Life Technologies) was added into the reaction mixture and the DNA was purified by Micro Bio-Spin 30 Column (Bio–Rad). The purified DNA was incubated with 0.5 µL M270 Streptavidin beads (Life Technologies) pre-blocked with salmon sperm DNA in buffer 1 (5 mM Tris pH 7.5, 0.5 mM EDTA, 1 M NaCl and 0.2% Tween 20) for 30 min. The beads were subsequently undergone three 5-min washes each with buffer 1, buffer 2 (buffer 1 without NaCl), buffer 3 (buffer 1 with pH 9) and buffer 4 (buffer 3 without NaCl). All binding and washing were done at room temperature with gentle rotation. Beads were then resuspended in water and amplified with 14 (cfDNA) or 9 (blood genomic DNA) cycles of PCR amplification using Phusion DNA polymerase (NEB). The PCR products were purified using AMPure XP beads. Separate input libraries were made by direct PCR from ligated DNA without labeling and capture. Pair-end 75 bp sequencing was performed on the NextSeq instrument.
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Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina NextSeq 500 |
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Description |
5hmC capture sequencing of cell free DNA
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Data processing |
bcl-fastq base-calling bowtie2 2.2.5 alignment samtools 0.1.19 filtering bedtools for reads counting and rpkm calculation N/A Genome_build: hg19 Supplementary_files_format_and_content: rpkm matrix
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Submission date |
May 11, 2016 |
Last update date |
May 15, 2019 |
Contact name |
Dan Xie |
E-mail(s) |
danxie@stanford.edu
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Organization name |
Sichuan University
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Department |
West China Hospital
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Lab |
State Key Laboratory of Biotherapy
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Street address |
#17 Renmin Road
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City |
Chengdu |
State/province |
Sichuan |
ZIP/Postal code |
610041 |
Country |
China |
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Platform ID |
GPL18573 |
Series (1) |
GSE81314 |
Shotgun sequencing of 5-hydroxymethylcysotine circulating cell-free DNA from the blood distinguishes solid tumor tissues of origin |
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Relations |
BioSample |
SAMN04961239 |
SRA |
SRX1748195 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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