DRP005281
16s rRNA sequencing of 1) intestinal fecal contents of surfactant D (SP-D)-deficient or wild type mice and 2) SP-D-binding or non-binding bacteria sorted from feces of wild type FVB mice.
For comparative analysis of microbiome between Sftpd-wt or ko mice (1)), fecal pellets from wt or ko mice were suspended in PBS and subjected to enzyme-based DNA extraction process. For analysis of SP-D-binding bacteria (2)), fecal pellets suspended in 1mM CaCl2/PBS were filtered through a strainer and the flow-throughs were centrifuged collect bacterial pellets. Fecal bacterial pellets were stained using anti-SP-D Ab and fluorochrome-conjugated secondary antibody. 2x106 of SP-D-positive or negative bacteria were collected by a cell sorter to be subjected to enzyme-based DNA extraction process. The 16S rRNA gene hypervariable V1-V2 regions were PCR-amplified using universal primers (a barcoded 27Fmod and 338R) and, multiplexed amplicon sequencing was carried out using MiSeq (illumina).