Total RNA was extracted from cells using RNeasy Micro Kit (QIAGEN 74004). For mRNA sequencing cDNA synthesis was performed using SMART-Seq Ultra Low Input RNA Kit for Sequencing (Takara 634889) from approximately 500 pg of total RNA. cDNA was validated using the High Sensitivity NGS Fragment Analysis Kit (Agilent formerly AATI DNF-474-0500) on a 12-Capillary Fragment Analyzer. Quantification was determined using the Quant-iT dsDNA Assay Kit, high sensitivity (Thermo Fisher Q33120), and 100 pg of cDNA was tagmented and ligated using the Nextera XT DNA Library Kit (Illumina FC-131-1024) at volumes to produce sequencing libraries. The resulting libraries were validated using the High Sensitivity NGS Fragment Analysis Kit on a 12-Capillary Fragment Analyzer and quantified using the Quant-iT dsDNA Assay Kit, high sensitivity. Equal concentrations of libraries were pooled, denatured, diluted, and subjected to paired-end sequencing using the Mid Output v2.5 kit (Illumina FC-404-2001) on a NextSeq550 following the manufacturer’s instructions.
Mid output v2.5 kit
Manufactured by Illumina
The Mid Output v2.5 kit is a laboratory equipment product from Illumina. It is designed to perform a specific function in the laboratory setting, but a detailed and unbiased description of its core function cannot be provided without the risk of extrapolation or interpretation. The details of this product's capabilities and intended use are best obtained directly from Illumina's product information.
Automatically generated - may contain errors
Lab products found in correlation
Nextera xt dna library kit, by Illumina (1 mentions)
High sensitivity ngs fragment analysis kit, by Agilent Technologies (1 mentions)
Smart seq ultra low input rna kit for sequencing, by Takara Bio (1 mentions)
Quant it dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Nextseq 500, by Illumina (1 mentions)
2 protocols using mid output v2.5 kit
1
Single-cell RNA-seq Library Preparation
2
Single-Cell RNA-Seq Library Preparation and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Libraries were prepared from isolated total RNA using the QIAseq UPX 3′ Transcriptome from Qiagen. Unique molecular identifiers and sample barcodes were introduced during first-strand synthesis, and all samples were pooled before second-strand synthesis and library preparation. The pooled libraries were paired end sequenced (124 × 28) on the NextSeq 500 using the Mid output V2.5 kit (Illumina). Read data were processed using the Qiagen GeneGlobe UPX 3′ Transcriptome primary and Secondary analysis tools. Read data were aligned to the human hg19 or mouse mm10 genome, and EdgeR (Robinson et al., 2010 (link)) outputs were used to call differentially expressed genes. Volcano plots were made using DEseq2 (Love et al., 2014 (link)) in Rosalind (Onramp Bio). GSEA was performed using the Broad Institute platform (v4.0.3; Subramanian et al., 2005 (link)). Gene sets of the hallmark gene sets, C2 curated gene sets, and C5 GO gene sets from the Molecular Signature Database collection were used for analysis with default settings. All RNA-seq data are available at Gene Expression Omnibus accession number GSE150875 .
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!