TITLE: DNA extraction from rectal mucosa biopsies and matched faecal samples taken from surgery for microbiome analysis
AUTHORS: Lee Murphy, Alan Maclean, Katarzyna Hafezi

[DESCRIPTION]
This protocol outlines the procedure for manual DNA extraction of microbiome DNA from rectal mucosa biopsy tissue and matched faecal samples taken during surgery and frozen at -80oC. 

The protocol covers
1. DNA extraction from faecal samples using MP Biomedicals FastDNA Spin Kit and FastPrep-24 for bead-beating
2. DNA extraction from rectal mucosa biopsy tissue using a modification of MP Biomedicals FastDNA Spin Kit and FastPrep-24 for bead-beating

Other steps not covered in this protocol are
3. Microbial DNA enrichment of extracted samples using NEB NEBNext Microbiome DNA Enrichment kit 
4. DNA Quantitation of extracted DNA using the Qubit Flourometer 
5. Library Preparation for microbiome WGS using NEB NEBNext Ultra II FS DNA Library Prep Kit for Illumina
6. DNA sequencing on the Illumina NextSeq2000

[BEFORE_START]
Ensure SEWSM buffer has 100% Ethanol added according to the kit instructions before use



[STEPS]
SECTION: DNA extraction from rectal mucosa tissue samples
21. Add 828 µL Sodium Phosphate buffer; 122 µL MT buffer (both from MPBio Kit), 50 µL Lysozyme (100mg/ml) and 50 µL Mutanolysin (5U/μl) to the tube, briefly vortex to mix and incubate on a heat block at 37 °C for 60 min 

SECTION: DNA extraction from rectal mucosa tissue samples
20. Label and weigh a 2ml Eppendorf tube. Transfer rectal mucosa biopsy tissue in to the 2ml Eppendorf tube. Re-weigh to calculate weight of biopsy tissue and record. (Aim for 5-60 mg of starting tissue).

SECTION: DNA extraction from rectal mucosa tissue samples
23. Perform Bead Beating with the MP Biomedicals FastPrep-24 for 1 min at 5m/s.

SECTION: DNA extraction from rectal mucosa tissue samples
22. Transfer entire contents of the Eppendorf tube to a Matrix E tube (MPBio Kit).
SECTION: DNA extraction from rectal mucosa tissue samples
24. Incubate at 85 °C  for 15 min on the heating block.

SECTION: DNA extraction from rectal mucosa tissue samples
25. Repeat steps 23 and 24.

SECTION: DNA extraction from rectal mucosa tissue samples
26. Centrifuge at 14000 rpm for  5 min and transfer supernatant to a fresh 2ml Eppendorf tube.

SECTION: DNA extraction from rectal mucosa tissue samples
27. Add 250 µL PPS Solution (MPBio), mix by inverting tube 12 times.

SECTION: DNA extraction from rectal mucosa tissue samples
28. Centrifuge at 14000 rpm for 5 min  and transfer supernatant to a 15ml tube. 
SECTION: DNA extraction from rectal mucosa tissue samples
33. Centrifuge at 14000 rpm for 1 min then empty the flow through from the tube.

SECTION: DNA extraction from rectal mucosa tissue samples
32. Gently resuspend the Matrix in the remaining solution by pipetting up and down and transfer 600 µL to a spin filter column (MPBio).

SECTION: DNA extraction from rectal mucosa tissue samples
31. Remove and discard 500 µL of supernatent without disturbing the Binding Matrix at the bottom of the tube.

SECTION: DNA extraction from rectal mucosa tissue samples
30. Mix by inverting the 15ml tubes for 2 min then leave standing for 5 min to collect the binding matrix at the bottom of the 15ml tube.

SECTION: DNA extraction from rectal mucosa tissue samples
34. Repeat steps 32 and 33 if necessary until all the Binding Matrix solution is applied to the spin filter column.

SECTION: DNA extraction from rectal mucosa tissue samples
39. Centrifuge 14k for 1 min  to collect eluted sample. Remove the spin column and store the DNA sample tube for QC and microbial DNA enrichment.

SECTION: DNA extraction from rectal mucosa tissue samples
38. Apply 80 µL DES elution buffer to the Matrix in the spin column and pipette gently up and down to resuspend the Matrix. (Heating the DES buffer on the block for 5 min  at 55 °C  before using may make this easier).

SECTION: DNA extraction from rectal mucosa tissue samples
37. Air dry for 5 min then transfer the spin column to a fresh, labelled catch tube (MPBio).

SECTION: DNA extraction from rectal mucosa tissue samples
36.  Centrifuge at 14000 rpm for 1 min. Discard the flow through and centrifuge at 14000 rpm for 2 min to dry.

SECTION: DNA extraction from rectal mucosa tissue samples
35. Add 500 µL SEWSM wash buffer (MPBio) to the spin column, pipette gently until the Matrix is fully resuspended. (Ensure SEWSM buffer has 100% Ethanol added according to the kit instructions before use)

SECTION: DNA extraction from rectal mucosa tissue samples
29. Vortex the Binding Matrix (MPBio) before use to ensure matrix is fully resuspended and add 1 mL to the 15ml tube.

SECTION: DNA extraction from faecal samples
1. Label and weigh a 2 mL  Matrix E tube tube (MPBio). Transfer stool (approximately 0.2 g ) and weigh to record weight of stool sample to be extracted.

SECTION: DNA extraction from faecal samples
2. Add 928 µL Sodium Phosphate buffer; 122 µL MT buffer (both from MPBio Kit) to the sample in the Matrix E tube.
SECTION: DNA extraction from faecal samples
3. Perform Bead Beating with the MP Biomedicals FastPrep-24 for 1 min at 5m/s.

SECTION: DNA extraction from faecal samples
4. Incubate at 85 °C  for 15 min on the heating block.

SECTION: DNA extraction from faecal samples
5. Repeat steps 3 and 4.
SECTION: DNA extraction from faecal samples
6. Centrifuge at 14000 rpm for  5 min and transfer supernatant to a fresh 2ml Eppendorf tube.

SECTION: DNA extraction from faecal samples
7. Add 250 µL PPS Solution (MPBio), mix by inverting tube 12 times.

SECTION: DNA extraction from faecal samples
8. Centrifuge at 14000 rpm for 5 min  and transfer supernatant to a 15ml tube. 
SECTION: DNA extraction from faecal samples
9. Vortex the Binding Matrix (MPBio) before use to ensure matrix is fully resuspended and add 1 mL to the 15ml tube.

SECTION: DNA extraction from faecal samples
10. Mix by inverting the 15ml tubes for 2 min then leave standing for 5 min to collect the binding matrix at the bottom of the 15ml tube.

SECTION: DNA extraction from faecal samples
11. Remove and discard 500 µL of supernatent without disturbing the Binding Matrix at the bottom of the tube.

SECTION: DNA extraction from faecal samples
12. Gently resuspend the Matrix in the remaining solution by pipetting up and down and transfer 600 µL to a spin filter column (MPBio).

SECTION: DNA extraction from faecal samples
13. Centrifuge at 14000 rpm for 1 min then empty the flow through from the tube.

SECTION: DNA extraction from faecal samples
14. Repeat steps 12 and 13 if necessary until all the Binding Matrix solution is applied to the spin filter column.

SECTION: DNA extraction from faecal samples
15. Add 500 µL SEWSM wash buffer (MPBio) to the spin column, pipette gently until the Matrix is fully resuspended. (Ensure SEWSM buffer has 100% Ethanol added according to the kit instructions before use)

SECTION: DNA extraction from faecal samples
16.  Centrifuge at 14000 rpm for 1 min. Discard the flow through and centrifuge at 14000 rpm for 2 min to dry.

SECTION: DNA extraction from faecal samples
17. Air dry for 5 min then transfer the spin column to a fresh, labelled catch tube (MPBio).

SECTION: DNA extraction from faecal samples
18. Apply 80 µL DES elution buffer to the Matrix in the spin column and pipette gently up and down to resuspend the Matrix. (Heating the DES buffer on the block for 5 min  at 55 °C  before using may make this easier).

SECTION: DNA extraction from faecal samples
19. Centrifuge 14k for 1 min  to collect eluted sample. Remove the spin column and store the DNA sample tube for QC.