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).

本研究中，为开展Sftpd野生型（Sftpd-wt）与敲除型（Sftpd-ko）小鼠的微生物组比较分析(1)，将野生型与敲除型小鼠的粪便颗粒悬浮于磷酸盐缓冲液（Phosphate Buffered Saline, PBS）中，随后进行酶法DNA提取。针对表面活性蛋白D（Surfactant Protein D, SP-D）结合菌的分析(2)，将粪便颗粒悬浮于1mM氯化钙-磷酸盐缓冲液（1mM CaCl₂/PBS）中，经滤器过滤后收集滤出液，通过离心获取细菌沉淀。将所得粪便细菌沉淀用抗SP-D抗体（anti-SP-D Ab）与荧光素偶联二抗染色，随后通过流式细胞分选仪收集2×10⁶个SP-D阳性与阴性细菌，再次进行酶法DNA提取。采用通用引物（带条形码标记的27Fmod与338R）对16S rRNA基因的高变V1-V2区进行PCR扩增，随后使用Illumina MiSeq测序平台开展多重扩增子测序。