Total RNA was extracted using a GenElute Mammalian Total RNA Miniprep Kit (Sigma) according to the manufacturer’s protocol. RNA samples were treated with RNase-free DNase (Sigma), and quality control was determined using a Qubit assay and an Agilent 2100 Bioanalyzer, as per each manufacturer’s specifications. Sample amounts were normalized and 1000 ng was used for library preparation using the NEB Ultra RNA Library Preparation Kit as per the manufacturer’s instructions. Library QC and quantitation were performed on all individual libraries using the Qubit assay and the Agilent 2100 Bioanalyzer. Libraries were normalized and pooled via Qubit measurement. The final pool was quantitated via qPCR. Sequencing was performed on the Illumina HiSeq 2500 Rapid Run Mode on one flowcell (two lanes) as per the system manufacturer. Raw RNASeq data were processed, normalized, and mapped to the human reference genome (hg19) using CLC Genomics Workbench 8 (Qiagen). Differentially expressed genes were identified using DESeq2 (35 (link)) with a significance cutoff of 0.001. Hierarchical clustering was performed using Cluster 3.0/Java TreeView.
Hiseq2500 rapid run mode
Manufactured by Illumina
Sourced in United States
The HiSeq2500 Rapid Run mode is a high-throughput sequencing system designed to deliver fast turnaround times for a range of sequencing applications. It provides rapid sequencing of DNA or RNA samples with a simple workflow.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Genelute mammalian total rna miniprep kit, by Merck Group (1 mentions)
Clc genomics workbench 8, by Qiagen (1 mentions)
Dnase free rnase, by Merck Group (1 mentions)
Casava v1, by Illumina (1 mentions)
Agilent sureselect xt, by Agilent Technologies (1 mentions)
Dna smart chip seq kit, by Takara Bio (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Nebnext library quant kit, by New England Biolabs (1 mentions)
2.0 fluorometer, by Agilent Technologies (1 mentions)
Seqcap ez choice library, by Roche (1 mentions)
Truseq small rna library preparation kit, by Illumina (1 mentions)
5 protocols using hiseq2500 rapid run mode
1
RNA-seq Library Preparation and Analysis
2
Multigene Panel Sequencing for Carrier Screening
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Inheritest Carrier Screen NGS tests are multigene panels that include the CBS gene. Library preparation and target enrichment were performed using the Agilent SureSelectXT method (Agilent Technologies, Santa Clara, CA). Briefly, genomic DNA was fragmented by sonication (Covaris, Woburn, MA), followed by end-repair, A-tailing and adaptor ligation. A target enrichment step was carried out using custom Agilent RNA probes, which were designed to capture all coding exons and flanking sequences of all isoforms for 144 genes (~ 0.5 M bases total), followed by post-capture indexing of the libraries by PCR for multiplex sequencing. Up to 96 libraries were pooled together and paired-end sequenced at 2 × 150 cycles using either the MiSeqV3 chemistry or the HiSeq2500 Rapid Run mode (Illumina, San Diego, CA) at 15 × minimum base coverage. Raw sequencing data were demultiplexed with the Illumina CASAVA v.1.8.2 software to generate fastq sequences for each sample.
3
ssDNA Library Preparation and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ssDNA samples prepared above were quantified using the Quibit ssDNA Assay Kit (molecular probes, life technologies), and 5–10 ng of each sample was used for library preparation. Libraries were prepared using DNA SMART ChIP-Seq Kit (Clontech) and Illumina indexed oligomers. Following PCR amplifications (15 cycles), libraries were size selected and purified using Agencourt AMPure XP beads (200–400 bp range). Each library was validated using Quibit 2.0 Fluorometer for concentration, Agilent 2100 Bioanalyzer for fragment size, and NEBNext Library Quant Kit (New England BioLabs) for adaptor presence. Typical library yield was 10–40 ng with average size of 400 bp. Finally, libraries from 12 samples were pooled in equimolar ratio (10 nM) and submitted for sequencing using Illumina HiSeq2500 Rapid Run mode (GENEWIZ, South Plainfield, NJ). A total of ~140 million 50 bp reads were obtained per pooled sample. The reads for each sample in the pool were separated per the corresponding Illumina indexes (% of >= Q30 bases: 97.85; mean quality score: 38.63), before subjecting to bioinformatics analysis.
4
Illumina TrueSeq DNA Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Libraries were prepared using 5 μg of genomic DNA according to the Illumina TrueSeq sample preparation protocol as described at http://support.illumina.com/content/dam/illumina-support/documents/myillumina/f5f619d3-2c4c-489b-80a3-e0414baa4e89/truseq_dna_sampleprep_guide_15026486_c.pdf (Illumina, San Diego, CA). After appropriate quality controls, libraries were pooled and captured using the SeqCap EZ Choice Library (Roche/NimbleGen, Madison, WI) following the manufacturer’s protocol, as detailed at the following http://www.nimblegen.com/products/lit/06560881001_SeqCapEZLibraryLR_Guide_v2p0.pdf . Captured libraries were multiplexed two-seven samples per lane and sequenced to generate 2x150 bp paired-end reads using the HiSeq2500 rapid run mode (Illumina, San Diego, CA).
5
Murine Cortex Small RNA Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA quality: The quality of total RNA extracted from 12 murine cortices was assessed using Agilent’s 4200 TapeStation (Agilent Technologies, Santa Clara, CA, USA), according to the manufacturer’s High Sensitivity Kit protocol. Samples with an RNA integrity number (RIN) of 7.5 or above were considered to be of good quality, and 8 of them (4 per group, NexKO or control) were chosen for small RNA sequencing.
Sequencing: A transcriptome library was constructed from all 8 samples using the Illumina TruSeq Small RNA Library Preparation Kit (Illumina, San Diego, CA, USA). Then, sequencing was performed using the Illumina (USA) HiSeq2500 Rapid Run mode with 50 bp single-end configuration, with 15.625 M reads per sample.
Analysis: RNA-Seq data were aligned and quantified according to the miRBase mouse assembly (mmu-21), using miR-MaGiC v1.0 [81 (link)]. DESeq2 1.24.0 [82 (link)] was used to normalize count data and calculate differential expression. miRNAs with FDR-adjusted p-values of 0.06 or less were considered to be differentially expressed. Experimentally verified miRNA target genes were obtained through multiMiR 1.10.0 [83 (link)] and tested for gene ontology enrichment with clusterProfiler 3.16.1 [84 (link)].
Sequencing: A transcriptome library was constructed from all 8 samples using the Illumina TruSeq Small RNA Library Preparation Kit (Illumina, San Diego, CA, USA). Then, sequencing was performed using the Illumina (USA) HiSeq2500 Rapid Run mode with 50 bp single-end configuration, with 15.625 M reads per sample.
Analysis: RNA-Seq data were aligned and quantified according to the miRBase mouse assembly (mmu-21), using miR-MaGiC v1.0 [81 (link)]. DESeq2 1.24.0 [82 (link)] was used to normalize count data and calculate differential expression. miRNAs with FDR-adjusted p-values of 0.06 or less were considered to be differentially expressed. Experimentally verified miRNA target genes were obtained through multiMiR 1.10.0 [83 (link)] and tested for gene ontology enrichment with clusterProfiler 3.16.1 [84 (link)].
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!