B4PC2 consortium metatranscriptomics
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.