KAPA Stranded mRNA-Seq Kit

RNA sequencing Mouse - Chondrocytes

Experiment
RNA sequencing Mouse - Chondrocytes
Product
KAPA Stranded mRNA-Seq Kit from Roche Lifesciences
Manufacturer
Roche Lifesciences

Protocol tips

Protocol tips
This protocol has been validated for library construction from 100 ng – 4 μg intact, total RNA, in ≤50 µL of RNase-free water.

Publication protocol

Primary chondrocytes of Col11a2-CreER; Sik3flox/flox mice were cultured in DMEM, 10% FBS medium for 5 days on a culture dish. Then, the chondrocytes were centrifuged and incubated in tubes for 3 days. The resulting cell pellets were incubated in the same medium in the presence or absence of 300 μM pterosin B for another 4 weeks. RNAs were extracted from the cultured chondrocyte pellets using QIAzol Lysis Reagent (Qiagen) and miRNeasy Mini Kit (Qiagen). RNAs were also extracted from various mouse tissues.
A 100 ng of total RNA was subjected to library preparation using KAPA stranded mRNA-seq Kits (KAPA Biosystems, Woburn, MA), according to the manufacturer's instruction. The libraries were sequenced in 100-cycle single-read mode of HiSeq2500. All sequence reads were extracted in FASTQ format using BCL2FASTQ Conversion Software 1.8.4 in the CASAVA 1.8.2 pipeline. The sequence reads were mapped to mm9 reference genes, using Tophat v2.0.14. Gene expression values were calculated and normalized using rpkmforgenes (10 December 2012). Averages of gene expression values from triplicate cultured chondrocyte pellet samples were calculated. Two-way hierarchical clustering of gene expressions in cultured chondrocyte pellets (n=3) and tissues (n=1) was conducted using the Euclidian distance and the ward method of hclust function in R3.2.1.

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Discussion

Discussion

5 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 - Chondrocytes using KAPA Stranded mRNA-Seq Kit from Roche Lifesciences.

Paper title
Pterosin B prevents chondrocyte hypertrophy and osteoarthritis in mice by inhibiting Sik3
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Manufacturer protocol

Download the product protocol from Roche Lifesciences for KAPA Stranded mRNA-Seq Kit below.

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