TruSeq Stranded Total RNA
RNA sequencing Mouse - RAW264.7
Publication protocol
Before RNA isolation, cells were washed by PBS for three times. Total RNA was isolated using TRIzol reagent (Invitrogen, USA). Samples were sent to Beijing Genomics Institute (BGI) for further bioinformatic analysis. BGI is a genome sequencing center headquartered in Shenzhen, Guangdong Province, China. RNA purity was assessed using the ND-1000 NanoDrop. The A260/A280 ratio for each RNA sample was greater than 1.8 and the A260/A230 ratio was greater than 2.0. RNA integrity was evaluated using the Agilent 2100 TapeStation. Only RNA samples with a RINe value above 7.0 were retained. Oligo(dT) magnetic beads were used to isolate poly(A) + mRNAs. The mRNAs were fragmented to approximately 200 bp in fragmentation buffer. Subsequently, these fragments were used as templates for first-strand and second-strand cDNA synthesis. The double-stranded cDNA fragments were purified, end-repaired, and then ligated to sequencing adapters. After purification, suitable fragments were enriched by PCR amplification according to instructions of TruSeq® RNA LT/HT Sample Prep Kit (Illumina). Finally, PCR products were purified and quantified for high-throughput sequencing using the Illumina HiSeq™ 2500.Full paper Login or join for free to view the full paper.
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Discussion
Discussion
4 years ago
Author:
Panagiotis Karagiannis
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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Manufacturer protocol
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