tag:blogger.com,1999:blog-32079676.post6428063144322440177..comments2024-02-13T21:22:02.522-08:00Comments on RRResearch: Planning more RNAseq experimentsRosie Redfieldhttp://www.blogger.com/profile/06807912674127645263noreply@blogger.comBlogger4125tag:blogger.com,1999:blog-32079676.post-35632383116615979332014-06-18T19:41:50.465-07:002014-06-18T19:41:50.465-07:00Sorry for the late reply, I've been "wrap...Sorry for the late reply, I've been "wrapping up" in the lab and am now furiously writing my thesis. No, Hfq is not a direct component of my thesis project. I have focused on Fur-regulated sRNAs (and discovered it, soon to be published). <br /><br />So I have only tangentially looked at Hfq in H. flu. To be honest, we have not found a true role for Hfq in NTHi 86-028NP. We cannot find a testable phenotype in an hfq mutant. Terry Stull published a paper on Hfq's role in virulence. Their lab was also unable to find any testable phenotypes in an hfq mutant like those seen for E. coli etc.<br /><br />However, I have evidence that Hfq is important in the regulation of a few genes suspected of being regulated by small RNAs. So I am a little skeptical on how big a role Hfq plays in H. flu gene regulation compared to other well-studied bacteria. Unfortunately, we have no data about sxy and sRNAs. Our lab's initial screen for small RNAs by RNA-seq was fairly short-sighted and only was able to identify a small RNA directly regulated by Fur (similar to RyhB in E. coli). However, we did grow NTHi 86-028NP in sBHI to mid-log and performed RNA-seq. We then annotated all potential small RNA loci. That data might complement whatever RNA-seq you have done as far as identifying potential sRNAs. That list will be published as suppl. table in my paper.<br /><br />My "selfish" reason for liking the idea of Hfq and sRNAs regulating competence comes from my project's focus on sRNAs in general. We once attempted an Hfq pulldown in NTHi 86-028NP but only recovered a very small list of RNAs bound to Hfq (but our result may have just been due to uz being novices at performing Hfq pulldowns). Im pretty positive after I graduate in August our lab will not be pursing sRNA/Hfq/competence work at all. <br /><br />Good luck! I am really interested in your work and the idea that (in the simplest terms) nutritional need drives many phenotypes in H. flu and bacteria in general. <br /><br />It's been great reading your blog the last 4 years or so. After I graduate I will be starting a fellowship in Sacramento, CA designed to get PhD scientists into science policy careers to advise policy makers on the science behind their policy issues. <br /><br />Im not sure how you get your RNA-seq data anaylzed, but Bob has a great collaboration with a bioinformatics group out of IUPUI in Indianapolis. They are smart and have a very fast turnaround. <br /><br />-Estevan Santana<br />Munson Lab<br />santana.7@osu.eduAnonymousnoreply@blogger.comtag:blogger.com,1999:blog-32079676.post-12767963234283763062014-03-17T07:58:55.132-07:002014-03-17T07:58:55.132-07:00We'll definitely be looking for small RNAs aff...We'll definitely be looking for small RNAs affecting sxy expression. There's also the toxin-antitoxin genes, which are the kind that affect RNAs.<br /><br />Do you have a 'selfish' interest in this because it's part of your thesis project? If so, might we collaborate rather than stepping on your toes?Rosie Redfieldhttps://www.blogger.com/profile/06807912674127645263noreply@blogger.comtag:blogger.com,1999:blog-32079676.post-20979024021597882122014-03-17T07:56:45.019-07:002014-03-17T07:56:45.019-07:00This comment has been removed by the author.Rosie Redfieldhttps://www.blogger.com/profile/06807912674127645263noreply@blogger.comtag:blogger.com,1999:blog-32079676.post-59491020995331732942014-02-14T10:42:42.096-08:002014-02-14T10:42:42.096-08:00For selfish reasons the hfq mutant is my choice (b...For selfish reasons the <i>hfq</i> mutant is my choice (but I think in some later posts you've already decided on the strains). My real question is does your RNA isolation method also collect the small RNAs? I think to fully rule out an sRNA role in competence development that information on sRNA expression is key. For some reason I still stubbornly like the idea of an sRNA influencing competence. <br /><br />-Estevan Santana<br />Munson Lab grad studentAnonymousnoreply@blogger.com