meth_4.1.11 Illumina TruSeq Stranded mRNA Library Preparation for Metatranscriptomics (Marine Microbes Project 2015-2017)

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

Library preparation

  1. Perform TruSeq Stranded mRNA-seq library preparation according the TruSeq Stranded mRNA Sample Preparation Guide (Document # 15031047 Rev. E, October 2013), skipping the purification of poly(A) RNAs.
  2. Add 13 µl of Fragment, Prime and Finish Mix to 5 µl of rRNA depleted rRNA sample and follow the library prep protocol from the Incubate RFP step (page 20).
  3. At the Enrich DNA Fragments step (page 38-41) perform 13-15 cycles of PCR

For inputs of 1 µg use 13 cycles, for inputs < 200 ng use 15 cycles

Quality check of the libraries

  1. Assess the size of the RNA-seq libraries via electrophoresis using the Agilent TapeStation TapeScreen DNA 1000 Assay or similar (Agilent Bioanalyzer, Perkin Elmer LabChip GXII).
  2. Quantify libraries using qPCR (KAPA Library Quantification Kits for Illumina or similar).
  3. Normalise the libraries and pool libraries for sequence following the library prep protocol (Normalise and Pool Libraries, page 44).