Environmental sampling of the METATRANSCRIPTOMIC for Subject
Libraries were prepared using Life Technologies Ion Total RNA seq kit v2 following ÒMAN0010654 Rev. B.0 - low input optionsÓ with the following modifications. The maximum of 10 ul of purified RNA was input into the fragmentation reaction with the 10-minute incubation time at 37¡C. ÒAssess the yield and size distribution of fragmented RNAÓ was omitted after purification of the fragmented RNA. Instead, all 12 ul of fragmented RNA was input into the hybridization reaction as indicated in the protocol for low input samples. ÊThe ligation reaction was incubated for 1 hour. The libraries were barcoded and amplified for 16 cycles. After the purification of the amplified cDNA according to protocol pages 16-28, one additional bead clean-up was performed with Agencourt Ampure XP beads. Molecular water was added to each sample to bring the volume to 30 ul and 30 ul of Ampure XP beads were added for a 1.0x bead clean up. This cleanup was performed according to protocol MAN009847 Rev. C.0 ÒIon Xpress Plus gDNA Fragment Library PreparationÓ. Templating and loading the chip were performed on the Ion Torrent Chef using Ion 540 Kit- Chef MAN0014119 Rev. C.0. Libraries were diluted to 50 pM and multiplexed at up to 4 samples per chip. All Chef runs were performed overnight with the ÒpauseÓ option to perform quality control of Ion Sphere Particle templating efficiency. Chips were sequenced the following day on the Ion Torrent S5 sequencer.
Subject-015-NP