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Sample GSM2150148

Query DataSets for GSM2150148

Status

Public on Jul 13, 2017

Title

gDNA extracted from healthy control 5 blood cells

Sample type

SRA

Source name

gDNA extracted from healthy control 5 blood cells

Organism

Homo sapiens

Characteristics

sample name: 36-blood
tissue: blood
target molecule: genomic DNA

Treatment protocol

not applicable

Growth protocol

not applicable

Extracted molecule

genomic DNA

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.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina NextSeq 500

Description

5hmC capture sequencing of cell free DNA

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

Submission date

May 11, 2016

Last update date

May 15, 2019

Contact name

Dan Xie

E-mail(s)

danxie@stanford.edu

Organization name

Sichuan University

Department

West China Hospital