Getting ready for RNA-seq cell/RNA preps

The RA's missing notebook hasn't turned up, so I don't have her notes of how she prepared the samples for the RNA-seq analysis. Luckily the main procedures are ones she used in many experiments and are described in her earlier notebooks and in an email she sent me. The basic procedures:Collecting samples:Grow cells to desired state in rich medium (sBHI) or competence medium (MIV).Mix 2 ml with 4 ml RNAprotect reagent (Qiagen); leave 5 min at RT.  We have 100 ml and can get more quickly through LSC Stores.Mix 2 x 1 ml with 0.25 ml 80% glycerol and freeze at -80°C. (for later competence assays).Pellet RNAprotect cells and freeze at -80°C.Preparing RNAs:Thaw cell pelletsUse Qiagen RNA prep kit.  We have lots and can get more quickly through LSC Stores.Don't use the DNase step.Elute in 40 µl H20.Measure concentration of 1 µl with Nanodrop.Run 4 µl in a 1% agarose TAE gel at 60V.Treat to remove DNA:Use volume containing 1 µg RNA (using RNA concentration from Nanodrop)Use Ambion Turbo DNase Use protocol in RA's notebook #1 (not missing); 2 x 30 min incubationsTreat to remove rRNA:Use Ribo-Zero kit to remove the rRNA from each sample.Samples:Here's the chart showing the MIV-competence samples I had planned.  I'm only going to do one set of the ∆hfq strain now, because our procedures aren't optimized for small RNAs (poor recovery and no strand information).  One of our summer-Honours students will be working on this mutant, and he can take my pre...
Source: RRResearch - Category: Medical Scientists Authors: Source Type: blogs