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Old 03-08-2016, 12:21 PM   #1
sallen
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Location: Falmouth, MA

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Default Secondary size selection for single cell RNAseq on NextSeq 500 help

Our lab is performing single cell RNAseq and mapping the reads back to an existing genome. We are running paired-end sequencing on a NextSeq 500 to analyze differential expression patterns between cell types. I am having difficulty deciding how narrow a range to size select for (using Pippin Prep) after adding adapter sequences. I am aware that tighter size selection reduces bias during bridge amplification, and that the bioinformatics are typically happier with narrower size ranges, but I want to make sure that my size distribution is broad enough to capture a representative sample. What size ranges are people using in their research? Do people feel that selecting transcripts in the 250-300 bp size range would be representative, or should I go tighter/broader? My Covaris sheared libraries currently span 50-1000 bp with the peaks around 300 bp. Any feedback is greatly appreciated!
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