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Old 10-13-2012, 08:03 AM   #1
wacguy
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Location: NC

Join Date: Sep 2012
Posts: 24
Default 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
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