RNA was extracted using Qiagen Allprep DNA/RNA/miRNA universal kit, which simultaneously isolates genomic DNA and total RNA. RNA concentration was evaluated using a NanoDrop spectrophotometer (ThermoFisher, Waltham, Massachusetts, USA) and RNA integrity was evaluated using the Bioanalyzer RNA 6000 Nano kit (Agilent Technologies, Santa Clara, California, USA). MiRNA NGS libraries were then prepared using TruSeq Small RNA Library protocol and sequences using HiSeq 2500 High Throughput Sequencer (all from Illumina, San Diego, California, USA).
Hiseq 2500 high throughput sequencer
Manufactured by Illumina
Sourced in United States
The HiSeq 2500 High Throughput Sequencer is a next-generation sequencing instrument manufactured by Illumina. It is designed to generate high-quality DNA sequencing data at a high throughput.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Bioanalyzer rna 6000 nano kit, by Agilent Technologies (1 mentions)
Allprep dna rna mirna universal kit, by Qiagen (1 mentions)
Clc genomic workbench v7.5, by Qiagen (1 mentions)
Nextera library preparation kit, by Illumina (1 mentions)
Masterpure dna purification kit, by Illumina (1 mentions)
Random hexamer primers, by Illumina (1 mentions)
Dnase 1, by Takara Bio (1 mentions)
Superscript double stranded cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyser, by Agilent Technologies (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Truseq rna sample preparation kit, by Illumina (1 mentions)
Nd 2000, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
7 protocols using hiseq 2500 high throughput sequencer
1
Comprehensive miRNA Sequencing Protocol
2
Small RNA-seq of Tumor Samples
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RNA from two tumor samples (before and after treatment) with high quality were used to prepare small RNA NGS libraries (RIN > 6), using TruSeq Small RNA Library preparation protocol. Successfully prepared libraries were sequenced using Illumina HiSeq 2500 High-Throughput Sequencer using single end sequencing (50 bp).
3′ adapter sequences were automatically identified and trimmed; reads were quality-filtered (Q 33) and reads within length of 18 and 27 nt were retained for mapping using sRNAbench (Rueda et al. 2015 (link)), fastx-toolkit and custom perl scripts. Reads were mapped to MirGeneDB (Fromm et al. 2015 (link)) using bowtie1.2 (Langmead et al. 2009 (link)), requiring an 18 nt seed sequence of zero mismatches to avoid cross-mapping. Mapped reads were counted using “summarizeOverlaps()” function from the “GenomicAlignments” Bioconductor package (Lawrence et al. 2013 (link)).
3′ adapter sequences were automatically identified and trimmed; reads were quality-filtered (Q 33) and reads within length of 18 and 27 nt were retained for mapping using sRNAbench (Rueda et al. 2015 (link)), fastx-toolkit and custom perl scripts. Reads were mapped to MirGeneDB (Fromm et al. 2015 (link)) using bowtie1.2 (Langmead et al. 2009 (link)), requiring an 18 nt seed sequence of zero mismatches to avoid cross-mapping. Mapped reads were counted using “summarizeOverlaps()” function from the “GenomicAlignments” Bioconductor package (Lawrence et al. 2013 (link)).
3
Genome Sequencing of Strain 863
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The genomic DNA from strain 863 was extracted using the Masterpure DNA Purification Kit (Epicentre, Madison, WI, USA) according to the manufacturer’s protocol. The quality of the genomic DNA was assessed using a Nanodrop Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The extracted DNA was used in Nextera Library Preparation Kit (Illumina San Diego, CA, USA). The prepared library was sequenced using 100 bp × 2 cartridge in HiSeq 2500 High Throughput Sequencer (Illumina) on rapid run mode. The quality of the sequenced data was assessed using FastQC software [22 ]. The sequenced data were then trimmed and assembled using CLC Genomic Workbench (V7.5; Qiagen, Hilden, Germany). Following this, the assembled sequence was annotated by the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Automatic Annotation Pipeline (PGAAP) [23 (link)] and RAST [24 (link)].
4
Total RNA Isolation and mRNA-seq Library Preparation
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Total RNA was isolated using TRIzol® Reagent (Invitrogen, CA, U.S.) according to the manufacturer’s instructions, and genomic DNA was removed using DNase I (Takara, Tokyo, Japan). Then RNA quality was determined with a 2100 Bioanalyser (Agilent, CA, U.S.) and quantified using a ND-2000 (NanoDrop Technologies, DE, U.S.). Three RNA samples from each group (Testis, PreO, Day1O, and Day6O) were pooled equally, and the mRNA-seq libraries were then prepared using 5 μg of pooled total RNA and the TruSeqTM RNA sample preparation Kit (Illumina, San Diego, CA). First, mRNA was isolated with oligo(dT) beads and then fragmented to 100–400 bp by fragmentation buffer. Second, double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA) with random hexamer primers (Illumina). Then the synthesized cDNA was subjected to end-repair, phosphorylation, and ‘A’ base addition according to Illumina’s library construction protocol. The libraries were amplified by PCR for 15 cycles using Phusion DNA polymerase (NEB), and then target cDNA fragments of 200–300 bp were selected using 2 % Low Range Ultra Agarose gel. After quantification by TBS380, paired-end sequencing of 101 bp reads was performed for the four cDNA libraries in one lane on an Illumina HiSeq2500 high-throughput sequencer.
5
Genome-wide shRNA Screening in Glioblastoma Stem Cells
6
GDNF Treatment on C6 Cells
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C6 cells were cultured and treated with GDNF for 0, 0.5, 1, and 24 h, respectively and total RNA was extracted from the four sample groups using Trizol reagent. All experiments were run in triplicate. After passing the electrophoretic quality inspection, performed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, US), total RNA was purified with a RNA clean XP kit and RNase free DNase set. Bidirectional RNA-Seq of the four samples were performed with an Illumina hiseq2500 high-throughput sequencer.
7
RNA-seq Analysis Pipeline
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Sequencing was performed on a HiSeq 2500 High Throughput Sequencer (Illumina). Single-end 50-mer reads were aligned using Tophat v2.1.1 (Kim et al., 2013) . Gene expression was determined using Cufflinks v2.2.1 and the FPKM (Fragments Per Kilobase Million) metric (Trapnell et al., 2010) .
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