title: GSM9512263: IgA-Seq Sample IgA2A; Homo sapiens; OTHER
library_construction_protocol: Frozen stool (200 mg) samples from each patient were thawed and dissolved in 1 mL of phosphate buffered saline (Invitrogen, Catalog No. Catalog No. AM9624). Stool was resuspended in 1 mL of 5 mM N-acetyl-cysteine (Thermo Scientific Chemicals, Catalog No. 616-91-1) to break disulfide bonds in mucus and release bacterial cells and then passed through 100 uM filters and centrifuged. The resulting stool pellets were resuspended in 1 mL of PBS and divided into four fractions for downstream experiments (bacterial FACS, whole microbiome 16S, IgA-Seq, and IgG-Seq). For bacterial FACS, stool cell pellets were centrifuged and resuspended in PBS and stained with 1:10,000 SYBR Green (Sigma Aldrich) at room temperature in dark for 20 minutes. Cell pellets were subsequently centrifuged, resuspended in PBS, and stained with 1:250 Anti-Human IgA-APC (REAfinity™, Catalog No. 130-116-879) and 1:250 of Anti-Human IgG-PE (REAfinity™, Catalog No. 130-119-878) and incubated on ice for 20 minutes. Cell pellets were centrifuged and resuspended in FACS buffer and 1:50 Fc block was to each sample and incubated at room temperature for 10 minutes. Bacterial cell pellets were then centrifuged and resuspended in FACS buffer. FACS was used to quantify the proportion of SYBR green positive (live bacterial cells) that were IgA-APC+, IgG-PE+, and IgA+IgG+ double positives.Stool pellets from each patient were processed as previously described. Bacterial cell pellets were resuspended in PBS and separate stool fractions were stained with either 1:250 Anti-Human IgA-APC (REAfinity™, Catalog No. 130-116-879) or 1:250 of Anti-Human IgG-PE (REAfinity™, Catalog No. 130-119-878) and incubated on ice for 20 minutes. Bacterial cell pellets were centrifuged and then washed with MACS buffer.  Anti-PE MicroBeads (Miltenyi Biotec, Catalog No. 130-090-855) were added to IgG fraction and Anti-APC MicroBeads (Miltenyi Biotec, Catalog No. 130-048-801) to IgA fraction. Bacterial cell pellets were then centrifuged, resuspended in MACS buffer, and placed onto OctoMACS™ Separator (Miltenyi Biotec, Catalog No. 130-042-108) columns for magnetic separation. IgA and IgG-bound bacterial fractions were then collected for each sample. Purity of IgA-bound and IgG-bound gut bacteria were above 90% for all samples included in downstream analyses. DNA from bacterial cell pellets from IgA-bound, IgG-bound, and unfractionated (whole gut microbiome) samples were isolated using the PureLink™ Microbiome DNA Purification Kit (Catalog No. A29790) per manufacturer's instructions. Bacterial 16S sequencing of the V4 and V5 regions (primer 515F/sequence GTGCCAGCMGCCGCGGTAA, primer 907R/sequence CCGTCAATTCCTTTGAGTTT) were performed at Novogene using 2x250 sequencing on an Illumina MiSeq.
identifiers.namespace: GEO
identifiers.id: GSM9512263_r1