Qiaamp genomic dna kit

DNA was extracted from tissue samples using the QIAamp Genomic DNA kit (Qiagen GmbH). The quantification and quality of the DNA were assessed using Qubit 2.0 fluorimeter with ds DNA HS assay kit (Thermo Fisher Scientific, Inc.) and the Agilent 2100 BioAnalyzer (Agilent Technologies, Inc.). Sequencing libraries were prepared based on the Illumina standard library construction instructions (Illumina, Inc.).

Genomic DNA of NSCs and CD133⁺ GBM CSCs was extracted and purified using QIAamp® genomic DNA kit (Qiagen, Catalog # 51304) following the manufacturer’s protocol. The concentration of gDNA was measured with the NanoDrop 8000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) at 260nm.

The sequence of pri-miR-181c-5p was amplified from the genomic DNA of human Peripheral Blood Mononuclear Cells (PBMCs), extracted by QIAamp genomic DNA kit (QIAGEN, USA), using Thermo Scientific DreamTaq Green PCR master mix (Thermo Fisher Scientific, Waltham, MA, USA) and forward primer: 5′-TCGA-GGATCC- ACTTAAGGAGCGGGCTTGAG-3′ and reverse primer: 5′-TCGA-GCTAGC- TCACAACCCACCGACAACA-3′, according to the manufacturer’s instruction. The PCR for pri-miR-181c-5p amplification was carried out as follows: 95 °C for 5 min; and 35 cycles of 94 °C for 15 s, 54 °C for 30 s, and 72 °C for 30 s. The amplified product was subsequently cloned in a pEGP-miR vector to form pmiR-181c-5p with the aid of Thermo Scientific T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA, USA). The cloned constructs were transformed subsequently into the cells of Escherichia coli TOP10 and confirmed by sequencing. Following the manufacturer’s guidelines, the verified clone was purified by an endotoxin-free GeneJET Plasmid Maxiprep Kit from Thermo Scientific (Thermo Fisher Scientific, Waltham, MA, USA). The Thermo Fisher Scientific Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the concentration and purity of the pmiR-181c-5p construct. The pEGP-miR null vector (pNull) was considered a control vector.

Tissue DNA was extracted utilizing the QIAamp Genomic DNA kit (Germany, QIAGEN) based on the manufacturer’s instructions. Sequencing libraries were generated according to Illumina standard library construction protocols (Illumina Inc.). The libraries were enriched using an Acornmed panel targeting 808 cancer-related genes. The captured libraries were then sequenced on the NovaSeq6000 System (Illumina Inc.). Sequencing reads were aligned to the reference human genome (hg19) using the BWA aligner (version 0.7.12). Base recalibration was conducted using GATK software (version 3.8). Single nucleotide variants (SNVs) and small insertions or deletions were identified using MuTect2 software (version 1.1.7). CONTRA software (version 2.0.8) was used to analyze copy number variant calling. An average coverage depth for tumor tissue was > 5000×. Mutations were identified based on these standards: mutant allele frequency (MAF) ≥ 1.0%, and at least 5 high-quality supporting reads (base quality ≥ 30, mapping quality ≥ 30).

Patient blood samples were collected at respective outpatient clinics at the Auckland, North Shore and Counties Manukau Hospitals, New Zealand. The blood samples of healthy controls were collected at the Faculty of Medical and Health Sciences, the University of Auckland, New Zealand and the New Zealand Blood Bank, Great South Road Centre, Epsom, Auckland New Zealand.
Blood samples from each participant were collected in Vacutainer
^® tubes (Becton Dickinson) containing EDTA by a trained phlebotomist. DNA was extracted using a QIAamp genomic DNA kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol, with the aid of a fully automated QIAcube (Qiagen, Hilden, Germany). The DNA samples were diluted to 5.0ng/μl as per requirements of the SEQUENOM MassARRAY iPLEX® assay protocol.