alias: GSM2809905
title: GSM2809905: HFD_peptide; feces metagenome; RNA-Seq
library_construction_protocol: Total bacterial RNA from feces sample was isolated with ZR Soil/Fecal RNA MicroPrep (Zymo research). Briefly, the sample was suspended in RNA lysis buffer and lysed by the mixer (Retsch). The supernatant was transferred to RNA-binding column and washed several times with RNA wash buffer. In-column DNaseI digestion at 25°C for 15 min was performed to eliminate DNA contamination in the sample. The presence of genomic DNA contamination was assessed by PCR with universal 16S rRNA gene primers. Before RNA-seq library preparation, rRNA was removed from 2 µg of total bacterial RNA with Ribo-Zero Bacteria kit (Illumina). 100 ng of purified RNA was used for RNA-seq library preparation and the cDNA was synthesized from reverse transcription with SuperScript III (Life technologies) and second-strand synthesis (New England Biolabs). The sequencing library was generated from purified cDNA with Nextera XT DNA library preparation kit (Illumina) and amplified by PCR. Cycling conditions were 72°C for 3 min, 95°C for 30 s, followed by 16 cycles of 95°C for 10 s, 55°C for 30 s, and 72°C for 30 s. A condition of 72°C for 5 min was used for the final elongation step. Libraries with different indexes were pooled and single-end sequencing (75bp) was performed on an Illumina NextSeq at the Scripps Research Institute next generation sequencing core.
identifiers.namespace: GEO
identifiers.id: GSM2809905