title: B4PC2 consortium metatranscriptomics
design: Total RNA was extracted from mouse cecum using the EZ1 RNA tissue kit (Qiagen, Germantown, MD) conducted on an EZ1 automated extraction instrument. Prior to extraction, tissue was homogenized with a FastPrep-24 5G bead-beating device (MP Biomedicals) at 6 m/s for 40 sec. Total RNA extracts subject to an additional DNase treatment using the RNeasy Mini Kit (Qiagen) based on an RNeasy Total RNA Cleanup Protocol (W.M. Keck, Foundation Biotechnology Resource Laboratory at Yale University). Microbial RNAseq libraries were prepared using the ScriptSeq v2 RNA-Seq Library Prep kit (Illumina, San Diego, CA). 250 ng of total RNA was prepared for library preparation by ribosomal RNA depletion using both mouse and bacterial Ribo-Zero rRNA Removal Kit (Illumina) according to the manufacturers instructions. The resulting double depleted RNA was used as input for ScriptSeq library preparation according to the manufacturers instructions.  Resulting libraries were pooled and sequenced on an Illumina NextSeq500 instrument using paired-end 2x150 base reads.