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Sample GSM4561856

Query DataSets for GSM4561856

Status

Public on Jul 15, 2020

Title

Normal-P1-22

Sample type

SRA

Source name

Plasma

Organism

Homo sapiens

Characteristics

sample type: Normal - Cancer-free

Growth protocol

Blood. tissue, and urine samples of healthy donors and cancer patients were collected and processed.

Extracted molecule

total RNA

Extraction protocol

Blood samples were allowed to clot at RT for 1-3 hours, centrifuged, processed as serum, filtered for cell contamination, and cryopreserved at -80°C . Aliquots of serum or plasma were thawed only once and mixed, before being analyzed for miR profiling by HTG WTA. Urine samples from healthy donors (n=8) and advanced stage melanoma patients’ (n=9) were collected and processed for miR NGS detection. Tumor tissues were obtained from elective surgeries performed at SJHC (D.K.). Formalin-fixed and paraffin-embedded (FFPE) tissues were obtained for HTG WTA
Libraries were prepared according to Illumina instrument sequencing protocols. Probe-captured miR samples were amplified and indexed via polymerase chain reaction (PCR) using master mix (dNTP 10 mM, 5X Hemo Klentaq® Reaction buffer, Hemo Klentaq® DNA polymerase (New England Biolabs Inc., Ipswich, MA), forward, and reverse primers (HTG Molecular Diagnostics Inc., Tucson, AZ).
Following PCR, library cleanup was performed using Ampure® XP beads (Beckman Coulter Inc., Brea, CA). Samples were mixed and incubated with the beads at RT for 5 minutes. The bead-bound libraries were washed twice with 80% ethanol to remove leftover PCR reagents and enzymes. After washing, the samples were dried at RT for 2 minutes to allow residual ethanol to evaporate. The libraries were eluted off the beads with 10 mM Tris-HCl, pH 8.0.

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 2500

Description

miRNA

Data processing

FASTQ files were generated from raw sequencing data using Illumina BaseSpace and Illumina Local Run Manager Software.
FASTQ files were analyzed with HTG EdgeSeq parser software to generate raw counts for 2,083 miRs per sample.
Supplementary_files_format_and_content: Raw counts obtained by using HTG EdgeSeq parser software

Submission date

May 20, 2020

Last update date

Mar 25, 2021

Contact name

Dave S.B. Hoon

E-mail(s)

[email protected]

Organization name

Saint John's Cancer Institute

Department

Translational Molecular Medicine