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Sample GSM1842541

Query DataSets for GSM1842541

Status

Public on Dec 31, 2020

Title

Plasma_exosomes_granuloma_1

Sample type

SRA

Source name

plasma exosome

Organism

Homo sapiens

Characteristics

tumor: granuloma

Growth protocol

EDTA Plasma samples stored at −80°C from 38 individuals with benign IPNs. Plasma from 7 early stage adenocarcinoma patients and 10 granuloma cases were used for biomarker discovery by next generation sequencing (NGS)

Extracted molecule

total RNA

Extraction protocol

50 µl serum was mixed well with 12.6 µl ExoQuick exosome isolation solution, and incubated on ice for 30 min. Then the samples were centrifuged at 1000 g for 30 min at 4°C. The pellet was washed wish 1X DPBS and resuspended in 50 µl 1X DPBS. 20 µl samples were loaded onto the nanochip and incubated at 37ºC for 2 h. On each chip, two wells with 1XDPBS were used as negative control.
The NextGen sequencing libraries were prepared using NEBNext Multiplex Small RNA Library Prep Set for Illumina sequencing kit (New England Biolabs). The 3’ and 5’ adapters and the reverse transcription primer were diluted in nuclease-free water to concentrations specified by the kit. The adapter sequences were ligated to the 3’ and 5’ ends of the RNA isolated from 100 μL of plasma using T4 RNA ligase. Ligation reactions were performed overnight at 16oC to maximize efficiency. cDNA was then transcribed from the ligation product followed by PCR amplification with a unique barcode sequence for each sample. The fragment size of about 150 nts, was then extracted and purified from an acrylamide gel. The final purified and barcoded libraries were then pooled in equal molar for single-end 50 nt sequencing run using an Illumina HiSeq2500 instrument.

Library strategy

ncRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 2500

Description

Plasma_exosomes_granuloma_DB343_L3_R1

Data processing

Basecalls performed using CASAVA version X.X.X
The reads were processed by trimming off the adaptor sequences and removing low quality sequences, polyA sequences and adaptor-only sequences. We used a “map and remove” strategy to map the sequence reads to various databases as described.
The processed sequences were first screened against known human microRNA, human transcripts, followed by human genomic sequence. To get mapping results, the alignment tool Blast was used to search microRNA and Bowtie was used to search other large databases.
For the endogenous sequence mapping, except microRNA, we applied three different levels of error tolerance: 0 mismatch, 1 and 2 mismatches. Due to the high sequence similarity for microRNA, we did not allow any sequence mismatch. After normalizing the mapping results with total number of reads, the number of reads mapped to a specific transcript should be proportional to its concentration in circulation.
Genome_build: miRBase21
Supplementary_files_format_and_content: tab-delimted text files include RPM values for each sample.

Submission date

Aug 03, 2015

Last update date

Dec 31, 2020

Contact name

Kai Wang

E-mail(s)

kwang@systemsbiology.org

Organization name

Institute for Systems Biology

Street address

401 Terry Ave N