metatranscriptome of sponge associated microbes
4ug of Qubit quantified total RNA was taken and subjected to rRNA depletion combining the RibominusTM Eukaryote Kit for RNA Seq (Invitrogen, Cat # A10837-08) and RibominusTM Bacteria Module (Invitrogen, Cat # K1550-04) protocols. The rRNA depletion was assessed by running an aliquot on RNA Nano Bioanalyzer chip.  Transcriptome library was constructed according to the NEXTflex RNA-Seq library protocol outlined in “NEXTflex RNA-Seq Sample Preparation Guide” (Cat # 5129-02). Briefly, 100ng ofQubit quantifiedrRNA depleted RNA was fragmented for 10 minutes at elevated temperature (95oC) in the presence of divalent cations and reverse transcribed with Superscript III Reverse transcriptase by priming with Random Hexamers.Second strand cDNA was synthesized using second strand synthesis mix. The cDNA was cleaned up using Agencourt AMPure XP SPRI beads (Beckman Coulter). NEXTflex™ RNA-Seq Barcode Adapters were ligated to the cDNA molecules after end repair and addition of “A”- base. SPRI cleanup was performed after ligation. The library was amplified using 12 cycles of PCR for enrichment of adapter ligated fragments. The prepared library was quantified using NanoDrop, Qubit and validated for quality by running an aliquot on High Sensitivity Bioanalyzer Chip (Agilent).