ena-STUDY-ROSWELL PARK CANCER INSTITUTE-03-01-2020-00:26:04:157-1619
Bacterial microbiomes of guts of male C57BL/6J mice optionally treated with metformin and/or diet to induce obesity
Briefly, male, 2-3-month old, C57BL/6J mice were treated with high-fat diet for 16 weeks to induce obesity. Age/sex-matched control mice were fed normal diet. Cohorts from obese and non-obese groups (continued on same diet) were injected subcutaneously with mouse Lewis lung carcinoma cells and subsequently monitored until established tumors were noted (~day 11), when mice were injected intra-peritoneally every two days with metformin (50 mg/kg in phosphate-buffered saline [PBS]) or PBS alone. Mice were sacrificed about 3 weeks after cell injection. Fecal pellets, produced by mice upon handling, were picked in 1.5-ml tubes, which after 2-3 hours were stored at -20 ºC for a few weeks followed by at -80 ºC for a few more weeks. Fecal pellets were collected from mice of two separate mouse experiments (expt. #2 or #3), immediately before start of metformin/PBS treatment ("pre") or immediately before mouse sacrifice ("post"). Fecal pellets were provided to Genomics facility of Roswell Park Comprehensive Cancer Center (RPCCC), Buffalo, USA. Subsequent work was performed at the facility in one batch. • DNA isolation & quantification: Qiagen® DNeasy PowerSoil Kit; single fecal pellet provided for each sample; elution with 100 µl; some were dried down in an attempt to get a quantifiable concentration; DNA quantified by Thermo® Qubit DNA High Sensitivity Kit. • 16S PCR and library preparation: As per Illumina® Metagenomics Guide, using the guide's suggested V3-V4 primers; Kapa Biosystems® KAPA HiFi ReadyMix for PCR, with 25 cycles and 25 ng DNA input (when available; otherwise entire DNA sample). Indexing with Nextera® XT v2 combinatorial dual index kit, 8 cycles of PCR. • Controls: one neg. ctrl. each for DNA extraction (empty tube) and 16S PCR (no template); one pos. ctrl. (ZymoBIOMICS™ Microbial Community DNA Standard, Zymo® cat. #6305). • Sequencing: all libraries together, at 8 pM each, with 10% PhiX library spike-in, on Illumina® MiSeq with v3 reagents, to obtain 300 b paired-end reads.
Briefly, male, 2-3-month old, C57BL/6J mice were treated with high-fat diet for 16 weeks to induce obesity. Age/sex-matched control mice were fed normal diet. Cohorts from obese and non-obese groups (continued on same diet) were injected subcutaneously with mouse Lewis lung carcinoma cells and subsequently monitored until established tumors were noted (~day 11), when mice were injected intra-peritoneally every two days with metformin (50 mg/kg in phosphate-buffered saline [PBS]) or PBS alone. Mice were sacrificed about 3 weeks after cell injection. Fecal pellets, produced by mice upon handling, were picked in 1.5-ml tubes, which after 2-3 hours were stored at -20 ºC for a few weeks followed by at -80 ºC for a few more weeks. Fecal pellets were collected from mice of two separate mouse experiments (expt. #2 or #3), immediately before start of metformin/PBS treatment ("pre") or immediately before mouse sacrifice ("post"). Fecal pellets were provided to Genomics facility of Roswell Park Comprehensive Cancer Center (RPCCC), Buffalo, USA. Subsequent work was performed at the facility in one batch. • DNA isolation & quantification: Qiagen® DNeasy PowerSoil Kit; single fecal pellet provided for each sample; elution with 100 µl; some were dried down in an attempt to get a quantifiable concentration; DNA quantified by Thermo® Qubit DNA High Sensitivity Kit. • 16S PCR and library preparation: As per Illumina® Metagenomics Guide, using the guide's suggested V3-V4 primers; Kapa Biosystems® KAPA HiFi ReadyMix for PCR, with 25 cycles and 25 ng DNA input (when available; otherwise entire DNA sample). Indexing with Nextera® XT v2 combinatorial dual index kit, 8 cycles of PCR. • Controls: one neg. ctrl. each for DNA extraction (empty tube) and 16S PCR (no template); one pos. ctrl. (ZymoBIOMICS™ Microbial Community DNA Standard, Zymo® cat. #6305). • Sequencing: all libraries together, at 8 pM each, with 10% PhiX library spike-in, on Illumina® MiSeq with v3 reagents, to obtain 300 b paired-end reads.