GSM1138293: BB16; human gut metagenome; RNA-Seq
Microbial cells were lysed by a bead beater (BioSpec Products), total RNA was extracted with phenol:chloroform:isoamyl alcohol (pH 4.5, 125:24:1, Ambion 9720), purified using MEGAClear columns (Ambion), and rRNA was depleted via subtractive hybridization (MICROBExpress, Ambion, in addition to custom depletion oligos). The presence of genomic DNA contamination was assessed by PCR with universal 16S rDNA primers. cDNA was synthesized using SuperScript II and random hexamers (Invitrogen), followed by second strand synthesis with RNaseH and E.coli DNA polymerase (New England Biolabs). Samples were prepared for sequencing with an Illumina HiSeq instrument after enzymatic fragmentation (NEBE6040L/M0348S). The size distribution of each library was quantified on an Agilent HS-DNA chip.