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Would any one please share conditions used for fragmenting bacterial mRNA? I am shooting for ~200 nt and starting mRNA conc. is ~75-100 ng/. I am using NEB Next mRNA library kit. They recommend 5 min at 94 C in Mg buffer. However this is for eukaryotic mRNA. I will appreciate if someone could share their success story using this kit with bacterial (E. coli/Salmonella/Bacillus ...) mRNA. I have depleted total RNA using Epicenters Ribo-Zero kit. Thanks everyone!
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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