Ovation® RNA-Seq System V2

RNA sequencing Mouse - C2C12

Experiment
RNA sequencing Mouse - C2C12
Product
Ovation® RNA-Seq System V2 from NuGEN
Manufacturer
NuGEN

Protocol tips

Protocol tips
Input Amounts 500 pg – 100 ng

Publication protocol

After isolation of total RNA and RPAD-prepared RNA using miRNeasy Mini Kit, ribosomal (r)RNA was removed by rRNA Depletion Nano kit (Qiagen), whereupon cDNA was prepared and amplified using the Ovation RNA-Seq System V2 (NuGEN) kit following the manufacturer's instructions. The amplified cDNA was fragmented using a Bioruptor (Diagenode) followed by ligation of adaptors using TruSeq ChiP Sample Preparation kit (Illumina, San Diego, CA, USA), and DNA fragments ranging 300–350 bp were isolated from a 2.5% agarose gel and subjected to 17 cycles of PCR amplification. A Bioanalyzer 2100 instrument was used to analyze the quality of the cDNA libraries, which were sequenced using an Illumina HiSeq 2500 instrument (and deposited in GSE92632).
For identification of circRNAs in the RNA-Seq, adapter contamination was removed from the raw fastq files and TopHat2 (v2.1.0) was used to align the sequences to the human genome (hg19) and mouse genome (mm9). The aligned reads were used to identify the body of circular RNA and residual linear RNA using Cufflinks (v2.2.1) and default parameters (with the exception of assembled transfrags supported by five or more reported fragments). Both known (Ensembl GRCh37 Release 82 for human and Ensembl mm9 Release 67 for mouse) and novel transcripts were identified. The reads which did not align to the genome were used to identify fusion junctions using TopHat2. The CIRCexplorer program (v1.10) was used with the fusion junctions obtained from TopHat2 using the identified transcripts from the previous step to identify both the circularizing junction and the spliced sequence of circRNAs in HeLa and C2C12 cells,

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Discussion

Discussion

4 years ago

Author: Panagiotis Karagiannis Greece

Using RNA from formalin fixed paraffin embedded tissue sections for sequencing

Hello there! I have some slides with mouse lung tumors and I would like to do rna sequencing. Will I be able to extract enough/good quality rna from RNA from formalin fixed paraffin embedded tissue sections for sequencing to be successful?

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Papers

Check out relevant papers found by Labettor's AI that are relevant for performing RNA sequencing Mouse - C2C12 using Ovation® RNA-Seq System V2 from NuGEN.

Paper title
High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs
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Manufacturer protocol

Download the product protocol from NuGEN for Ovation® RNA-Seq System V2 below.

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Videos

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