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Sample GSM2384542

Query DataSets for GSM2384542

Status

Public on Sep 05, 2017

Title

MAZPL000030HEA

Sample type

SRA

Source name

blood

Organism

Homo sapiens

Characteristics

individual_id: MAZ000030HEA
age: 62
gender (1=male, 2=female): 1
disease site: healthy
disease state: healthy
tissue: plasma

Treatment protocol

Plasma sample was prepared within 6h by centrifuging twice at 1,350g for 12min, and then centrifuging at 13,500g for 12min. The prepared plasma samples (about 2ml/subject) were immediately stored at -80°C. About 10-25 mg tissue was collected using a scalpel after sample unfreezing.

Growth protocol

A total of 4ml of peripheral blood was collected from subject individuals using EDTA anticoagulant tubes. Tissue samples, including tumor and adjacent tissue samples, from patients were stocked at -80°C after surgical operation.

Extracted molecule

genomic DNA

Extraction protocol

The plasma cfDNA was isolated using the QIAamp Circulating Nucleic Acid Kit (Qiagen) according to the manufacturer’s protocol. Genomic DNA from tissues was isolated using the ZR Genomic DNA-Tissue Kits (Zymo Research) according to the manufacturer’s protocol.
First, the genomic DNA is fragmented using an enzymatic reaction. Next, the fragmented genomic DNA or the cfDNA were repaired and installed with the Illumina compatible adaptors. The glucosylation reactions were performed in a 25 μL solution containing 50 mM HEPES buffer (pH 8.0), 25 mM MgCl2, purified DNA, 100 μM N3-UDP-Glc, and 1 μM βGT, at 37°C for 1 hr. The reaction was purified by Micro Bio-Spin 30 Column (Bio–Rad) into ddH2O. After that, 1 μL DBCO-PEG4-DBCO (Click Chemistry Tools, 4.5 mM stock in DMSO) was added to the reaction mixture. The reactions were incubated at 37°C for 2 hr. Next, the DNA was purified by Micro Bio-Spin 30 Column (Bio-–Rad). The purified DNA was incubated with 5 µL C1 Streptavidin beads (Life Technologies) in 2X buffer (1X buffer: 5 mM Tris pH 7.5, 0.5 mM EDTA, 1 M NaCl) for 15 min according to the manufacturer’s instruction. The beads were subsequently washed eight times for 5 -min with 1X buffer. All binding and washing was done at room temperature with gentle rotation. The captured DNA fragments were amplified with 14-16 cycles of PCR amplification. The PCR products were purified using AMPure XP beads according to the manufacturer’s instructions. DNA concentration of each library was measured with a Qubit fluorometer (Life Technologies) and sequencing was performed on the Illumina Hi-Seq platform or NextSeq 500 platform.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Description

cell free DNA
Processed_Data
1856

Data processing

Read-through sequences within raw sequencing reads were trimmed using Trimmomatic version 0.35. Low quality bases at the 5' (Phred quality score <5) and 3' (5bp-sliding window Phred score< 15) were also trimmed. Reads with a minimum length of 50bp were aligned to the human genome assembly GRCh37 using Bowtie2 version 2.2.6 with end-to-end alignment mode. For paired-end sequencing data, read pairs were concordantly aligned with fragment length <500bp and with up to 1 ambiguous base and four mismatched bases per 100bp length.
Raw sequencing reads profiled for 5mC were trimmed and aligned to GRCh37 using Bismark version 0.15.0 according to previously published data processing procedures.
Alignments with Mapping Quality Score (MAPQ) ≥10 were counted for overlap with genomic features using featureCounts of subread version 1.5.0-p1, without strand information.
Gene regions were defined as the regions between gene start sites and gene end sites annotated by GENCODE release 19.
CpG islands were annotated CpG islands by UCSC Table Browser plus +/- 1kb regions.
Genome_build: GRCh37
Supplementary_files_format_and_content: Plain text format. Processed_Data is a data frame with fields: 1) gene (official gene symbol; for 23 pairs of genes sharing the same official gene symbol, one from each pair assigned a new gene symbol as original_gene_symbol.dup); 2) pos (chr_start_stop) of genes; 3) raw counts of samples at genes. Reports hydroxy-methylation levels.
Supplementary_files_format_and_content: Plain text format. Processed_5mc_Data is a data frame with fields: 1) pos (chr_start_stop) of CpG islands; 2) raw counts of samples at CpG islands. Reports methylation levels.

Submission date

Nov 05, 2016

Last update date

May 15, 2019

Contact name

Xu Zhang

E-mail(s)

xuzhangchicago@gmail.com

Organization name

University of Illinois at Chicago

Street address

820 South Wood St