title: GSM1185808: VKR_8.3V73_14dpc; mouse gut metagenome; RNA-Seq
library_construction_protocol: RNA was treated with RNAse-free TURBO-DNAse (Ambion) and 5S rRNA and tRNAs were removed (MEGAClear columns, Ambion). A second DNAse treatment was performed (Baseline-ZERO DNAse; Epicenter). rRNA was initially depleted using MICROBexpress kit (Ambion) followed by a second MEGAClear purification. In addition, custom biotinylated oligonucleotides, directed against conserved regions of sequenced human gut bacterial rRNA genes were employed for streptavidin bead-based pulldowns. cDNA was synthesized using SuperScript II (Invitrogen), followed by second strand synthesis with RNAseH, E. coli DNA polymerase (NEB) and E. coli DNA ligase (NEB). Samples were sheared using a BioRuptor XL sonicator (Diagenode); 150-200 bp fragments were gel selected and prepared for sequencing. Multiplex microbial cDNA sequencing was performed using an Illumina Hi-Seq2000 instrument to generate 23.7 ± 16.4 million unidirectional 101 nt reads per sample.