The PTEN mutation status of TCGA patients and the patients from our center was detected using whole-exome sequencing (WES). The tumor specimen that represents the characteristic was selected by experienced neurosurgeons for detection. Genomic DNA was extracted from fresh frozen tumor specimens and blood samples with a DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). WES libraries were prepared using Agilent’s SureSelect Human All Exon V5 Kit (Agilent Technologies, Santa Clara, CA, USA) and sequenced on the Illumina HiSeq2000 Genome Analyzer platform (Illumina, San Diego, CA, USA). Sequencing reads were aligned to a human reference genome (UCSC hg19) using the Burrows–Wheeler Aligner (BWA) (26 (link)). Subsequent processing was performed using PICARD (http://picard.sourceforge.net ), the Genome Analysis Toolkit (GATK), and VarScan 2 (27 (link)).
Hiseq 2000 genome analyzer platform
Manufactured by Illumina
Sourced in United States
The HiSeq 2000 Genome Analyzer platform is a high-throughput DNA sequencing system developed by Illumina. It is designed to perform massively parallel DNA sequencing, generating large volumes of sequence data. The core function of the HiSeq 2000 is to provide researchers with a tool for efficient and accurate DNA sequencing, enabling the analysis of genetic information.
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Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (6 mentions)
Rnase free dnase 1, by Thermo Fisher Scientific (2 mentions)
Sureselect human all exon v5 kit, by Agilent Technologies (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Rneasy ffpe kit, by Qiagen (1 mentions)
Truseq rna sample preparation kit, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Mrna seq library preparation kit, by Illumina (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Agilent bioanalyzer dna 2000 chip, by Agilent Technologies (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Rnaqueous kit, by Thermo Fisher Scientific (1 mentions)
Truseq sample preparation kit v2, by Illumina (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
7 protocols using hiseq 2000 genome analyzer platform
1
PTEN Mutation Detection by WES
2
Transcriptome Sequencing of Haemonchus contortus
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Our preliminary transcriptome sequencing of the ISE strain was carried out using both female and male samples (one sample per sex only). Standard Illumina TruSeq RNA libraries were prepared and transcriptome sequencing was carried out by GATC Biotech on an Illumina HiSeq2000 Genome Analyzer platform, using single read (1 × 100 bp) technology, yielding approximately 25 million reads for each of the female and male samples. Quality control measures and filtering of all RNA-Seq samples were carried out using CLC Genomics Workbench v8.1 (http://www.clcbio.com ) as previously described (Vogel et al., 2014 (link)). Digital gene expression analysis was carried out with CLC Genomics Workbench v8.1 to generate BAM mapping files, using the predicted coding sequence dataset of the H. contortus BioProject PRJNA205202 from the WormBase ParaSite website as a reference, and by counting the sequences to estimate expression levels, using previously described parameters for read mapping and normalization (Jacobs et al., 2016 (link)). An annotation file was generated using BLAST, Gene Ontology and InterProScan searches implemented in BLAST2GO PRO v2.6.1 (www.blast2go.de ). The data are presented as normalized log2-transformed RPKM values (normalizing across the samples, using as calculation the total number of mapped reads and the length of the reference sequence).
3
Transcriptome Profiling of Metastatic Tumor
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Using the delineation line drawn by the pathologist on the reverse side of each slice as a guide, the metastatic tumor in the SLN was scraped into a 1.5 ml RNase-free tube and sent for RNA extraction using the RNeasy FFPE kit (Qiagen, Germany) according to the manufacturer’s instructions. The obtained total RNA was measured using a NanoDrop 2000 (Thermo Scientific, USA) and stored at −80 °C until used. Libraries of mRNA derived from total RNA were constructed using the Illumina ®TruSeq™ RNA Sample Preparation Kit (USA) according to the manufacturer’s instructions. The concentration and size distribution of the libraries were determined using an Agilent 2100 Bioanalyzer (USA). The libraries were then sequenced using an Illumina Hiseq 2000 Genome Analyzer platform in paired-end 100-bp mode.
4
RNA-Seq Analysis of A549 Cells
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To prepare samples for RNA-Seq, we cultured A549 and A549/Abr cells in medium with or without 100 nM Abraxane for 4 h. In RNA-seq data analysis, we labeled these four differently treated cells as A549, A549/Abr, A549-100 and A549/Abr-100, respectively. Total RNA was extracted from these cells using TRIzol reagent (Invitrogen) as per the manufacturer’s instructions. RNA concentrations were determined using NanoDrop 2000 (Thermo Scientific). The integrity of RNA samples was determined using 1.2% agarose gel electrophoresis, followed by removal of the residual genomic DNA with RNase-free DNase I (Ambion). Using the Illumina mRNA-Seq library preparation kit, the cDNA library was constructed according to the manufacturer’s instructions. Transcriptome sequencing was then conducted using Illumina Hiseq 2000 Genome Analyzer platform in pair-ended manner with the read length of 100-bp.
5
Zebrafish RNA-Seq and miRNA-Seq
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Approximately 50 zebrafish embryos at 48 hpf from different experimental replicates were snap-frozen in liquid nitrogen. The total RNA was extracted using TRIzol (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions. The integrity of RNA samples was determined using 1.2% agarose gel electrophoresis, followed by removal of the residual genomic DNA with RNase-free DNaseI (Ambion, Austin, Texas, USA). mRNA and small RNA libraries were constructed using the Illumina mRNA-Seq and miRNA-seq library preparation kits, respectively. The size distribution and concentration of the libraries were determined by Agilent Bioanalyzer DNA 2000 chip (Agilent Technologies, Santa Clara, California, USA) followed by sequencing on the Illumina Hiseq 2000 Genome Analyzer platform. The RNA-Seq library was sequenced with 2 × 100 bp in pair-end mode by 100-bp lengths, and the miRNA library was sequenced in single-end mode by 80-bp lengths.
6
Total RNA Extraction and RNA-seq
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Total RNA was isolated from frozen tissues using a RNAqueous kit (Life Technologies, Carlsland, CA, USA). Short-insert ‘paired-end’ libraries were prepared using the Illumina TruSeq Sample Preparation Kit v2 and Illumina HiSeq 2000 next-generation sequencing was performed following the manufacturer's instructions (Illumina, San Diego, CA, USA). Libraries were sequenced bi-directionally (101 bp in each direction) on the Illumina HiSeq 2000 Genome Analyzer platform.
7
Genome-wide SNP detection pipeline
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Genomic DNA was extracted from 100 BC1 individuals using the classical phenol/chloroform method50 . The quality of DNA was checked with a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA), and through 1% agarose gel electrophoresis. DNA from each individual was digested with the restriction endonuclease EcoR I. Solexa P1 adapters were added to the digested fragments. The ligated DNA samples were pooled and sheared using a Covaris S-Series ultrasonicator (Covaris) to an average size of 500 bp. The sheared samples were separated in a 1.5% agarose gel, and DNA fragments of 300 to 700 bp were isolated and enzymatically blunted. The Klenow fragment (exo-; New England Biolabs) was then used to add an adenine to the 3′ end. An adapter with divergent ends (P2 adapter) was ligated to conduct selective PCR. The libraries were run for sequencing of paired-end reads (100 bp) on the Illumina HiSeq. 2000 Genome Analyzer platform (Novogene, Beijing, China).
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