Generally, iPS neurons were fractionated in a very similar method as the CAD cells. However, since the iPS neurons usually contained less material, instead of combining six wells together into one prep as was done for the CAD cells, between 6 and 12 wells were combined together. After mechanical fractionation by scraping into soma and neurite fractions and RNA isolation using the Zymo Quick RNA Microprep Kit, between 100 and 500 ng of neurite RNA was obtained.

Fractionation efficiency was monitored by protein dot blot using the beta-actin and histone H3 antibodies described above. PolyA-selected, stranded RNAseq libraries were constructed using the Kapa mRNA Hyperprep kit, 100 ng of total RNA, and 15 PCR cycles of library amplification. Each condition (FXS and unaffected; soma and neurite) was fractionated, prepared, and sequenced with four replicates for a total of 16 samples. Samples were sequenced using paired-end, 150 nt sequencing on an Illumina NovaSeq. Approximately 30 million read pairs were obtained per sample.

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Q&A
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Agnes Nishimura
King's College London 
Hello, I am trying to extract RNA from neurites and perform qPCR and I found your paper. Would you share your detailed protocol in how to isolate RNA from soma and neurite fractions please?

Many thanks!

Agnes
2020-09-23 03:11:52 Reply



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