Long term stability of laboratory mouse gut microbiota
Samples were transferred to sterile bead beading vials containing 0.7g zirconium beads, and 200l of 20% SDS, 282l Buffer A, 268l Buffer PM, 550l phenol/chloroform/isoamylalcohol (25:24:1, pH 8) were added. The pelleted cells were subject to phenol-chloroform-based extraction by (Rius, Kittelmann et al. 2012) that combined mechanical and chemical lysis of cells. The samples were quantified and diluted to 40 ng/l per sample and stored in -20 C for further use. The extracted DNA was processed according to the Earth Microbiome Project (http://www.earthmicrobiome.org/protocols-and-standards/16s/) as described in Thompson et al [13] except a dual-indexing strategy was used as described by (Fadrosh, Ma et al. 2014) to reduce the number of barcoded primers required. PCR was conducted with QIAGEN Taq MasterMix (CAT NO 201445, QIAGEN, Germany) in triplicates (plus one negative control per sample) under the condition: 94 C for 3 mins; 35 cycles of 94 C for 45 secs, 50 C for 60 C, 72 C for 90 secs; 72 C for 10 mins; 4 C hold. The amplicons were quantified with Quant IT Picogreen (CAT NO P7589, Thermo Fisher Scientific, USA), pooled together in equimolar concentrations and sequenced at Singapore Centre for Environmental Life Sciences Engineering (SCELSE) at Nanyang Technological University using Illumina MiSeq paired-end chemistry (251x251bp).
SUB4690875
3.H8_13417_48