meth_4.1.10 Illumina Ribo-Zero rRNA Removal Kit for Metatranscriptomics (Marine Microbes Project 2015-2017)

Contributor(s): Ramaciotti Centre for Genomics, UNSW, Sydney

This protocol describes the procedure for preparing metatranscriptomic libraries form samples to be sequenced on the Illumina HiSeq 2500 platform. The library preparation follows the Ribo-Zero rRNA Removal Kit Reference Guide (Document # 15066012 v02, August 2016) and TruSeq Stranded mRNA Sample Preparation Guide (Document # 15031047 Rev. E, October 2013) with some protocol variations.

Quality check of the input DNA

  1. Verify the quality of the extracted RNA using the Bioanalyzer RNA 6000 Nano or RNA 6000 Pico Kit.

Library preparation

Note: The Ribo-Zero rRNA Removal kits support rRNA depletion from 1-5 µg of total RNA. Probes from the Bacteria and Pant Ribo-Zero kits can be combined (50:50 ratio) to simultaneously deplete both bacterial and plant rRNA. In this instance the inputs of RNA should be halved (i.e., 500 ng -2.5 ng of total RNA). The inputs for the sediment RNA sample maybe much lower than these recommended inputs.

  1. Bring ≤1 µg of total RNA to a final volume of 28 µL with RNase-free water.
  2. Perform rRNA depletion following the Ribo-Zero Kit Reference Guide (Document # 15066012 v02, August 2016).
  3. Perform clean-up of rRNA depleted supernatant using Agencourt RNAClean XP Kit according to the Ribo-Zero Kit Reference Guide. Elute samples in 8.5 µL of RNase-free water.