TITLE: IgA Metagenomic Immunoglobulin Sequencing (MIG-Seq)
AUTHORS: Matthew R Olm, Sean Spencer

[DESCRIPTION]
IgA, the most highly produced human antibody, is continually secreted into the gut to shape the intestinal microbiota. Methodological limitations have critically hindered defining which microbial strains are targeted by IgA and why. Here, we develop a new technique, Metagenomic Immunoglobulin Sequencing (MIG-Seq), and use it to determine IgA coating levels for thousands of gut microbiome strains in healthy humans. We find that microbes associated with both health and disease have higher levels of coating, and that microbial genes are highly predictive of IgA binding levels, with mucus degradation genes especially correlated with high binding. We find a significant reduction in replication rates among microbes bound by IgA, and demonstrate that IgA binding is more correlated with host immune status than traditional microbial abundance measures. This study introduces a powerful technique for assessing strain-level IgA binding in human stool, paving the way for deeper understanding of IgA-based host microbe interactions.

[STEPS]
SECTION: Preparation of fecal samples
1. Weigh out 300 mg of thawed   in 2 mL centrifuge tube

SECTION: Preparation of fecal samples
3. Rehydrate sample with 1.25 mL cold  . Incubate 5 min on ice 

SECTION: Preparation of fecal samples
2. Vigorously vortex and mix samples via pipetting to ensure homogenization
SECTION: Preparation of fecal samples
4. Centrifuge samples at 500 x g, 15 min 

SECTION: Preparation of fecal samples
5. Following centrifugation, filter the clarified supernatant (~1mL) through a 70 μm filter
SECTION: Preparation of fecal samples
6. Remove and retain 100 µL of filtered supernatent for use as the "unsorted" or "native" fraction for subsequent metagenomic sequencing. Keep this fraction on ice for remainder of protocol.

SECTION: Preparation of fecal samples
7. Divide the remaining supernatant (~900 uL) into 2 separate wells of a 96 deep-well plate. These wells will be treated as individual samples throughout the remainder of the protocol, and the two samples will combined at the end of the protocol to increase sample biomass. 
SECTION: Antibody staining
10. Centrifuge the 96 deep-well plate at 5000 x g, 5 min

SECTION: Antibody staining
9. Prepare the following antibody master-mix in the staining buffer:
1:30 dilution of   
1:50 dilution of   
SECTION: Antibody staining
8. Prepare the following staining buffer in PBS:
3% Fetal Bovine Serum
0.5% Sodium Azide
1 mM EDTA

Keep staining buffer on ice
SECTION: Antibody staining
11. Discard supernatant and resuspend pellets in 100 µL antibody master-mix. Incubate on ice for 30 min 

SECTION: Antibody staining
12. Wash samples with 1 mL cold staining buffer. Centrifuge 5000 x g, 5 min 

SECTION: Antibody staining
13. Discard supernatent and resuspend pellet in 200 µL  staining buffer

SECTION: Antibody staining
14. Retain 20 µL of the resuspend pellet to act as a stained, unsorted control for Flow Cytometry analysis to establish background to calculate “Native IgA binding percentage”

SECTION: Magnetic cell separation
15. Stain the remaining 180 µL with 20 µL of EasySep APC Selection Cocktail  for 15 min at room tempterature

SECTION: Magnetic cell separation
16. Add 22 µL of EasySep Dextran RapidSpheres 50100   and incubate for 10 min at room temperature

SECTION: Magnetic cell separation
17. Place samples on 
 
 for 5 min  to allow for magnetic separation.

SECTION: Magnetic cell separation
18. Carefully discard unbound supernatent while on the magnet. Then remove 96-well plate from magnet and wash with 200 µL of staining buffer.

SECTION: Magnetic cell separation
19. Repeat steps 17 and 18 an additional 2 times, for a total of 3 5 min magnetic enrichments and washes.

SECTION: Magnetic cell separation
20. After the final resuspension, combine the duplicate wells (established in Step 7) to establish the "IgA positive" fraction
SECTION: Flow cytometry
21. Prepare tubes for flow cytometry by combining 5 µL of sample, 20 µL of counting beads, and a 1:10,000 dilution of    into staining buffer for a final volume of200 µL
SECTION: Flow cytometry
21.1. Tubes should be made using 1) unstained native sample (step 6), 2) stained native sample (from step 14), and 3) stained IgA+ samples (from step 20)

SECTION: Flow cytometry
22. Analyze cells on a flow cytometer
 

SECTION: Flow cytometry
22.1. Refer to this image to establish flow cytometry gates: 
 


SECTION: Flow cytometry
23. For each sample, the native “IgA fraction” is calculated as the number of IgA+ cells in the native sample (b) minus the number of IgA+ cells in the unstained control (a). Samples in which the unstained control (a) hadsa higher number of IgA+ cells than the native stained sample (b), or the native stained sample (b) had a higher number of IgA+ cells than the IgA+ sample (c), should be considered to have “failed QC” and be removed from subsequent analysis.