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We would like to use Illumina's TruSeq kit to prepare libraries for paired end RNA-seq. I would like to shoot for a longer insert size and am considering trying fragmentation with the Covaris method or a nebulizer. Any suggestions, advice, protocols, guidance, links would be greatly appreciated. Also - what size do you aim for?
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We tried all different conditions that they recommended for longer sizes and none worked well.
I would suggest creating full length cDNA from your RNA using other methods than the kit (SSIII for example) and then use a covaris to shear to the length you want. We have customers that do this very well. -
Hi Kwaraska,
Can you give me more information about the protocol used by your customers?
Can you describe the steps until you get cDNA from your non-fragmented RNA?Comment
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For cDNA conversion we use the Invitrogen products SSIII and second strand kit. We just follow the protocols.
Then we follow the Covaris recommended protocols for the different sizes. There is a sheet on their website that gives you conditions for all different sizes. On cDNA they work well and true to size.Comment
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Thank you very much kwaraska for your answer. We are also interested in preparing paired end Truseq stranded RNA seq with 200-300 bp inserts . In its protocol Illumina used dUTP for cDNA conversion and inserts are shorter than 200bp. Could someone give me advice how to proceed?Comment
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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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