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wacguy 10-13-2012 08:03 AM

library prep and...
 
Hi all,

I'd like to make a library from a small sample size (a few hundred cells) and I'd like to keep strandedness, and to get whole RNA (ie, not using poly(T) but random oligos). After a month and a half of digging, I've set my mind on Trizol prep>DNase treatment (Qiagen)>Ribozero depletion (Epicentre) and library preparation (ScriptSeq v2 RNA-Seq Library Preparation Kit, Epicentre). I'm not sure I will have enough RNA for the depletion and I was thinking, is there any reason not to start with library prep that also involves amplification and then go for rRNA depletion? Also, two technical question; for precipitation of RNA, NH4Ac or NaAc? How do I make the Ammonium acetate RNA-free, Sambrook and other protocols say not to autoclave and any filtration will not get rid of RNase.

Thank you very much everyone and have a great weekend,
Guy

Bitar 11-06-2012 12:43 PM

Sigma makes an RNAse free Ammonium Acetate, 7.5M (A2706-100ml) which is what I use in my RNA preps. Works well.
As for your other question, not sure RiboZero would work on cDNA (double stranded, DNA vs RNA, etc).


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