Shotgun metagenome sequencing of contents from the ileocecal junction: C57BL/6 J mice
Contents from the ileocecal junction (30 mg) mixed with 3.5 mL of Microbial Lysis Buffer 1, 100 L of Proteinase K as well as 30 L of RNase A followed by upside-down mixing multiple times. After incubating at 37C for 30 mins, add 1.2 mL of Microbial Lysis Buffer 2 followed by upside-down mixing. The reaction was incubated at 55C for 120 mins and then centrifuged for 10 mins. Transfered the obtained supernatant and run through Genomic-tip columns to purification genomic DNA. Adding the desired amount of EB buffer into sample tubes depending on the size of precipitated DNA. Incubate at 37C for 30-60 mins until DNA samples are completely dissolved for further nucleic acid quality control step. Genomic DNA was used with Illuminas TruSeq for library preparation. Libraries were pooled, and paired-end sequencing was conducted on an Illumina HiSeq PE 150 Platform