Sequencing bacterial mRNA with Illumina stranded kits

Noa · 11-12-2012, 12:13 PM

I am interested in directionally sequencing mRNA from bacteria (who do not have a polyA tail). Since Illumina's Ribo-zero kit depletes only mammalian rRNA I will not be able to use it. Therefore, I want to use my own bacterial Ribo-Zero kit and then use the Illumina stranded (directional) mRNA sequencing kit.

The main difference I see between the Ribo-Zero total RNA stranded kit and the mRNA stranded kit (apart from the mRNA/total RNA isolation procedure) is in the buffers used for fragmentation (FPF buffer for mRNA, EFP buffer for total RNA). Can the FPF buffer of the directional mRNA seq kit be used for fragmentation in solution rather than on the beads?

pmiguel · 11-14-2012, 04:13 AM

I don't know the answer to your specific question, but we frequently use ribo-zero with the TruSeq RNA prep kit by skipping the polyA step and adding 10-50ng of depleted RNA to the fragmentation step.

Call Illumina Tech support. Unlike most companies they actually tend to be able to provide useful answers.

--
Phillip

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11-12-2012, 12:13 PM	#1
Noa Member Location: haifa israel Join Date: Jun 2011 Posts: 62	Sequencing bacterial mRNA with Illumina stranded kits I am interested in directionally sequencing mRNA from bacteria (who do not have a polyA tail). Since Illumina's Ribo-zero kit depletes only mammalian rRNA I will not be able to use it. Therefore, I want to use my own bacterial Ribo-Zero kit and then use the Illumina stranded (directional) mRNA sequencing kit. The main difference I see between the Ribo-Zero total RNA stranded kit and the mRNA stranded kit (apart from the mRNA/total RNA isolation procedure) is in the buffers used for fragmentation (FPF buffer for mRNA, EFP buffer for total RNA). Can the FPF buffer of the directional mRNA seq kit be used for fragmentation in solution rather than on the beads?

11-14-2012, 04:13 AM	#2
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	I don't know the answer to your specific question, but we frequently use ribo-zero with the TruSeq RNA prep kit by skipping the polyA step and adding 10-50ng of depleted RNA to the fragmentation step. Call Illumina Tech support. Unlike most companies they actually tend to be able to provide useful answers. -- Phillip