Hi All,
I'm new to the forum so I'm sorry if I missed similar questions asked about low RNA input for library prep.
I'm trying to do expression analysis on around 10k mouse bone marrow cells, where I can purify about 0.5-3ng of total RNA using RNeasy plus micro kit in 12ul of RNase water. The bioanalyzer results look good for all samples with RIN>8.
Our lab used to use the Nugen Ovation RNA-seq system V2 to RT and amplify cDNA (>500pg according to the kit), but the problem is over-representation of rRNA reads in the final sequencing results. Also it seems a lot of waste when I amplify to get micrograms of cDNA and only use 100ng for the library prep (according to the next library prep kit from Nugen).
I recently found a kit from Clontech (SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian) that requires only 250pg-10ng total RNA input to generate sequencing directly, without amplification steps in between cDNA and adaptor ligation (only 5 cycles for adaptor PCR ligation). It seems to be a more cost-effective option and I should in principle avoid the amplification bias. I'm wondering if anyone has experience with this kit or how it is compared to the Nugen kit.
Thanks!
I'm new to the forum so I'm sorry if I missed similar questions asked about low RNA input for library prep.
I'm trying to do expression analysis on around 10k mouse bone marrow cells, where I can purify about 0.5-3ng of total RNA using RNeasy plus micro kit in 12ul of RNase water. The bioanalyzer results look good for all samples with RIN>8.
Our lab used to use the Nugen Ovation RNA-seq system V2 to RT and amplify cDNA (>500pg according to the kit), but the problem is over-representation of rRNA reads in the final sequencing results. Also it seems a lot of waste when I amplify to get micrograms of cDNA and only use 100ng for the library prep (according to the next library prep kit from Nugen).
I recently found a kit from Clontech (SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian) that requires only 250pg-10ng total RNA input to generate sequencing directly, without amplification steps in between cDNA and adaptor ligation (only 5 cycles for adaptor PCR ligation). It seems to be a more cost-effective option and I should in principle avoid the amplification bias. I'm wondering if anyone has experience with this kit or how it is compared to the Nugen kit.
Thanks!
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