The total RNA was isolated from the tissue samples using TRIzol reagent (Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. Then we assessed the RNA integrity and DNA contamination by using electrophoresis on a denaturing agarose gel. After confirming the RNA is intact and pure, we using the Ribo-Zero rRNA Removal Kit (Illumina San Diego, CA, USA) and the CircRNA Enrichment Kit (Cloud-seq USA) to remove the rRNA and enrich the circRNAs. The RNA-seq libraries were constructed by using pretreated RNAs with TruSeq Stranded Total RNA Library Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. The libraries were denatured as single-stranded DNA molecules, captured on Illumina flow cells, amplified in situ as clusters and finally sequenced for 150 cycles on Illumina HiSeq™ 4000 Sequencer (Illumina, San Diego, CA, USA according to the manufacturer's instructions. Paired-end reads were harvested from Illumina HiSeq™ 4000 Sequencer, and were quality controlled by Q30. The reads were aligned to the reference genome/transcriptome with STAR software and circRNAs were detected and annotated with DCC software. CircBase database and circ2Trait disease database were used to annotated the identified circRNAs. The differentially expressed circRNAs between the two groups were identified T test statistical methods.
Hiseq 4000 sequencer
Manufactured by Illumina
Sourced in United States, China, France, United Kingdom, Germany
The HiSeq 4000 is a high-throughput DNA sequencing system manufactured by Illumina. It is designed to generate large volumes of sequencing data at a rapid pace. The HiSeq 4000 uses Illumina's proprietary sequencing-by-synthesis technology to determine the nucleotide sequence of DNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (39 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (35 mentions)
Ribo zero rrna removal kit, by Illumina (29 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (26 mentions)
2100 bioanalyzer, by Agilent Technologies (25 mentions)
Truseq stranded total rna library prep kit, by Illumina (23 mentions)
Rneasy mini kit, by Qiagen (23 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (15 mentions)
Trizol, by Thermo Fisher Scientific (12 mentions)
Truseq stranded mrna kit, by Illumina (11 mentions)
Quantseq fwd kit, by Lexogen (11 mentions)
Tapestation, by Agilent Technologies (10 mentions)
Truseq stranded mrna library prep kit, by Illumina (9 mentions)
Nebnext ultra 2 directional rna library prep kit, by New England Biolabs (9 mentions)
Hiseq 2500 sequencer, by Illumina (9 mentions)
High sensitivity dna chip, by Agilent Technologies (9 mentions)
Agencourt ampure xp bead, by Beckman Coulter (8 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (7 mentions)
Kapa library quant kit, by Roche (7 mentions)
Labchip gx system, by PerkinElmer (7 mentions)
521 protocols using hiseq 4000 sequencer
1
Comprehensive Circular RNA Profiling
2
Genomic and Transcriptomic Profiling of Tumors
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Genomic DNA isolation and bulk DNA sequencing were performed as we described in our previous work [45 (link)]. Briefly, fresh tumors were surgically resected from these two patients. Each tissue was cut into two pieces, with one for further single-cell collection and the other for bulk sequencing. This procedure could maximally ensure that the single-cell and bulk sequencing data were generated from a close region of the tissue. Genomic DNA were extracted using the QIAamp DNA Mini Kit (QIAGEN). Exon libraries were constructed using the SureSelectXT Human All Exon V5 capture library (Agilent). Samples were sequenced on the Illumina Hiseq 4000 sequencer with 150-bp paired-end reads.
For bulk RNA analysis, small fragments of tumor tissues were first stored in RNAlater RNA stabilization reagent (QIAGEN) after surgical resection and kept on ice to avoid RNA degradation. RNA of tumor samples were extracted using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s specification. Libraries were constructed using NEBNext Poly (A) mRNA Magnetic Isolation Module kit (NEB) and NEBNext Ultra RNA Library Prep Kit for Illumina Paired-end Multiplexed Sequencing Library (NEB). Samples were sequenced on the Illumina Hiseq 4000 sequencer with 150-bp paired-end reads.
For bulk RNA analysis, small fragments of tumor tissues were first stored in RNAlater RNA stabilization reagent (QIAGEN) after surgical resection and kept on ice to avoid RNA degradation. RNA of tumor samples were extracted using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s specification. Libraries were constructed using NEBNext Poly (A) mRNA Magnetic Isolation Module kit (NEB) and NEBNext Ultra RNA Library Prep Kit for Illumina Paired-end Multiplexed Sequencing Library (NEB). Samples were sequenced on the Illumina Hiseq 4000 sequencer with 150-bp paired-end reads.
3
Ribosome Profiling and Total RNA-Seq of HeLa S3 Cells
4
Illumina-based Circular RNA Profiling
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After sequencing with the Illumina Hi Seq 4000 sequencer, the raw double-stranded reads were harvested after quality filtering. Raw paired-end reads were harvested from the Illumina Hi Seq 4000 sequencer after quality filtering. First, Q30 was used to carry out quality control. Cutadapt software (v1.9.3) was used to strip headers, and low-quality reads for high-quality reads were obtained. Then, the high-quality reads were aligned to the reference genome or transcriptome using BWA-MEM software (v.0.7.12), and circRNA detection and characterization were conducted with CIRI software (v.1.2) [28 (link)]. CircRNAs were identified by the CircBase database and circ2Trait disease database [29 (link),30 (link)], and novel circRNAs were labelled “novel”. The raw junction reads for all the samples were normalized by the total reads and the converted by log2. Fold changes ≥ 2.0 and p ≤ 0.05 were categorized as significantly differentially expressed circRNAs.
5
Ribosome Profiling and RNA-Seq of HeLa Cells
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HeLa S3 cells were grown in 10-cm dishes at 70-80% confluency and treated with SSA (100 ng/ml) or its solvent MeOH for 6 h before lysis. The libraries for ribosome profiling were prepared as described earlier 67, 82 and sequenced by a HiSeq4000 sequencer (Illumina).
Total RNA was extracted from the same lysate used for ribosome profiling with TRIzol LS (Thermo Fisher Scientific) and Direct-zol RNA MicroPrep Kits (Zymo Research). The libraries were prepared with the TruSeq Stranded mRNA Library Prep Kit followed by rRNA removal with Ribo-Zero Gold (Illumina) and were sequenced on a HiSeq4000 sequencer (Illumina).
Total RNA was extracted from the same lysate used for ribosome profiling with TRIzol LS (Thermo Fisher Scientific) and Direct-zol RNA MicroPrep Kits (Zymo Research). The libraries were prepared with the TruSeq Stranded mRNA Library Prep Kit followed by rRNA removal with Ribo-Zero Gold (Illumina) and were sequenced on a HiSeq4000 sequencer (Illumina).
6
Circularome Profiling via RNA-seq
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Briefly, paired‐end reads were gained from Illumina HiSeqTM 4000 sequencer (Illumina). After 3ʹ adaptor‐trimming and low‐quality reads were cut out by the cutadapt software (v1.9.3), the high‐quality reads were aligned to the reference genome using STAR software (v2.5.1b).29 Next, circRNAs were detected and annotated with the DCC software and the circBase database, respectively. The edgeR software (v3.16.5) was employed to normalized the data and performed for differentially expressed circRNA analysis.30
7
Differential RNA-seq analysis of tumor samples
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For all tumor samples, the RNA-seq libraries were constructed by using total RNA prepared with TruSeq RNA Exome (Illumina). The libraries were subsequently sequenced on Illumina HiSeqTM 4000 Sequencer (Illumina) according to the manufacturer’s instructions. The reads were aligned to the reference human genome (hg38) with STAR-2.7.1a software. The reads aligned to rRNA and transfer RNA (tRNA) regions were removed. The mapped reads were assigned to genes (Ensembl database annotation version GRCh38.98) using FeatureCount from the Bioconductor package Rsubread. The resulting FeatureCounts were then normalized as either transcripts per million (TPM) or fragments per kilobase million (FPKM) by R package neuMatIdx. Differential expression analysis was performed using DESeq2 (Love et al., 2014 (link)).
8
Small RNA Sequencing Library Preparation
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The RNA with a range of 18–30 nt in size was concentrated by polyacrylamide gel electrophoresis. Subsequently, the 3′ adapters were inserted into RNA, and enrichment was then performed for the RNA with 36–44 nt in length. The 5′ adapters were then ligated to the RNAs as well. The products were reverse transcribed by reverse transcription-polymerase chain reaction (RT-PCR), and the PCR products with 140–160 bp in size were used to generate cDNA libraries. Agilent 2100 and RT-quantitative PCR (RT-qPCR) were carried out to assess the libraries’ quality. The small RNA-seq was finally carried out using an Illumina HiSeqTM4000 sequencer (Illumina, San Diego, CA, USA).
9
RNA Extraction and Sequencing of T. mongolica
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T. mongolica leaves (one sample per population) were used for RNA isolation. Total RNA was extracted using a Plant Plus RNA Kit (DP437, Tiangen, Beijing, China), according to the manufacturer’s instructions. The extracted RNA was treated with deoxyribonuclease I (TaKaRa Bio Inc., Otsu, Shiga, Japan) for 30 min at 37 °C to remove residual DNA. RNA quality was monitored using an Agilent 2100 Bioanalyzer with a minimum RNA integrity number value of 8.0. Six cDNA libraries (D1–D6) were constructed and sequenced on a paired-end flow cell device using an Illumina HiSeqTM 4000 sequencer at Biomarker Biotechnology Co., Ltd (Beijing, China). The sequencing data were called and quality controlled through the Illumina data processing pipeline.
10
Transcriptome Analysis of Adipose Tissue
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Total RNA of adipose tissue was collected to analyze the transcriptome. A cDNA library was constructed using qualified adipose tissue RNA samples. The transcriptome sequencing processes of adipose tissue were performed by Novogene (Beijing, China) using Illumina HiSeqTM 4000 sequencer. The adaptors of paired-end reads were trimmed and quality control checks were carried out using trim-galore (v.0.6.0). Reads were aligned to mouse genome reference (GRCm38.p6) from GENCODE using STAR (v.2.7.3a). FeatureCounts (v.1.6.3) counted reads stored in BAM format to exon sites of genes included in GTF files from GENCODE. Differential gene expression analysis was conducted using raw counts as input using R (v.3.5.1) package DESeq2 (v.1.22.2). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Gene ontology (GO) enrichment analysis of differential genes were performed by R (v.3.5.1) package clusterProfiler (v3.10.1).
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