TITLE: RNA Extraction from Cecum Contents of Gnotobiotic Mice 
AUTHORS: Haley Gause

[DESCRIPTION]
RNA extraction from the cecum contents of gnotobiotic mice colonized with extremely minimal communities (single yeast and/or single bacterial community) can be difficult due to excess complex carbohydrates. These carbohydrates are not broken down in the same way as they are in conventional mice and their presence can significantly decrease yield and purity of your RNA sample. In this protocol, special considerations are taken, including extra phenol/chloroform extractions and lithium chloride precipitations, to reduce the contamination of carbohydrates. RNA resulting from this protocol is extremely clean (A260/280 = > 2.0, A260/230 = 2.0-2.3). 

[BEFORE_START]
Germ-free Mice have been pre-colonized via gavage with distinct gnotobiotic community of yeast and/or bacteria. 

[GUIDELINES]
This protocol is designed for RNA extraction from the cecum contents of gnotobiotic mice colonized with few (1-2) species, specifically colonized with Candida albicans and/or Enterococcus faecalis. 

[STEPS]
SECTION: At time of euthanization
1. Harvest cecum and cecum contents. Drop ½ of cecum contents into 10 ml RNAlater. Leaves tubes at 4oC for 1 day minimum (7 day maximum).
SECTION: At time of euthanization
2. Pre-weigh 2ml tubes. Add 1ml of RNAlater-cecum contents to each tube (~200mg contents) using wide-bore 1000µL tips and centrifuge at 3500RPM for 5 min to pellet contents. Remove supernatant. 

SECTION: At time of euthanization
3. Weigh pellets. Subtract weight of empty tube to find weight of cecum pellet.  Store pellet aliquots at -80oC.
SECTION: RNA Extraction
4. To 2ml RNase-free screw cap tube, add ~500µL ice-cold 0.5µM zirconia beads (1 tube per 100mg cecum contents). Keep tubes on ice until use.
SECTION: RNA Extraction
5. Place cecum contents aliquots on ice to thaw.
SECTION: RNA Extraction
6. Re-suspend aliquot in the following per 100mg (200mg – multiple all volumes by 2):
500µL Lysis Buffer (200 mM NaCl, 20mM EDTA)
210µL 20% SDS
500µL phenol:chloroform:isoamyl alcohol (PCI) (pH 4.5, 125:24:1)
SECTION: RNA Extraction
7. Transfer resuspended cecum contents to screw-cap tube containing beads. Split total volume between 2 bead-beating tubes (each has ~1210uL)
SECTION: RNA Extraction
8. Bead-beat tubes 5min using Mini-beadbeater
SECTION: RNA Extraction
9. Centrifuge @ 10000xg for 5 minutes to separate phases
SECTION: RNA Extraction
10. Transfer aqueous phase to new Eppendorf tube. Add equal volumes phenol:chloroform:isoamyl alcohol (PCI) and vortex 20s. Spin @ 10000xg for 5 minutes.
SECTION: RNA Extraction
11. Transfer aqueous phase to phase-lock tube (heavy). Add equal volume of PCI, shake well to mix and spin @ 10000xg for 3 minutes.
SECTION: RNA Extraction
12. Add equal volume PCI to aqueous above the phase-lock layer, mix well by shaking and spin @ 10000xg for 3 minutes.
SECTION: RNA Extraction
13.    two more times (total of 6 Phenol chloroform extractions). Move aqueous to new phase-lock tube as needed. 

SECTION: Isopropanol Precipitation
14. Decant aqueous phase from phase-lock tube to new RNase-free 1.5ml eppendorf tube. Add 1 volume RT isopropanol and place at -20C for 20 minutes.
Note: Any amount of time over 20 minutes will cause increased crashing out of the complex carbohydrates present and will reduce yield and purity. 
SECTION: Isopropanol Precipitation
15. Centrifuge at max speed for 25 min, 4oC. You will see a disk of RNA form between two layers (not a pellet like traditional precipitations).
SECTION: Isopropanol Precipitation
16. Carefully remove ALL liquid from tube (above and below disk of nucleic acid). If you don’t remove the liquid below the disk, the carbohydrates will begin to crash out and crystalize when doing the EtOH washes and your sample will be less pure.
SECTION: Isopropanol Precipitation
17. Wash 2X with 750µl 75% ethanol. Invert to wash tube. Centrifuge max speed for 5 minutes, 4oC. 
SECTION: Isopropanol Precipitation
18. Remove all ethanol from pellet (remove supernatant, quick spin tube, remove residual ethanol with aspirator). Air dry pellet for <5 min if a lot of residual ethanol remains.
SECTION: Isopropanol Precipitation
19. Resuspend pellet in 50 µL 45oC nuclease-free water
SECTION: Isopropanol Precipitation
19.1. Make sure pellet is completely resuspended before moving forward! Place at 45oC for a few minutes to fully resuspend
SECTION: NEB Monarch RNA Clean-up (500ug) (T2050)
20. Follow NEB Monarch RNA Clean-up as documented in product's protocol and detailed below: 
SECTION: NEB Monarch RNA Clean-up (500ug) (T2050)
21. Add 2X RNA Cleanup Buffer (100ul to 50ul sample)
SECTION: NEB Monarch RNA Clean-up (500ug) (T2050)
22. Add 1 vol 100% ethanol to sample (150ul)
SECTION: NEB Monarch RNA Clean-up (500ug) (T2050)
23. Invert/flick to mix.  Transfer to Spin-cartridge. Spin at 12000xg for 1min. Discard flow-through
SECTION: NEB Monarch RNA Clean-up (500ug) (T2050)
24. Add 700µL wash buffer I. Centrifuge and discard flow-through
SECTION: NEB Monarch RNA Clean-up (500ug) (T2050)
25. Wash 2X with 500µL wash buffer II. Centrifuge and discard flow-through.
SECTION: NEB Monarch RNA Clean-up (500ug) (T2050)
26. Centrifuge spin-cartridge ~ 12000xg for 1 min to dry column and membrane.
SECTION: NEB Monarch RNA Clean-up (500ug) (T2050)
27. Place dried column in clean Eppendorf tube.
SECTION: NEB Monarch RNA Clean-up (500ug) (T2050)
28. Add 50ul nuclease-free water to center of membrane. Let incubate at RT for 1 min. Centrifuge to collect flow-through.

SECTION: TURBO DNase Treatment
29. To 50µL sample, add and mix gently:  5 µL 10X buffer; 1 µl TURBO DNA
SECTION: TURBO DNase Treatment
30. Incubate at 37oC for 20-30 minutes
SECTION: TURBO DNase Treatment
31. Resuspend DNAse Inactivation Reagent by vortexing. Add 6 µL inactivation reagent to samples and mix by flicking.
33. Incubate at RT for 5 minutes. Flick tube occasionally to resuspend inactivation reagent.
32. Centrifuge tube @ 10000xg for 1.5 minutes. Carefully transfer 50ul into new tube.
SECTION: Lithium Chloride Precipitation
34. Add 25ul 7.5M Lithium chloride precipitation solution (final= 2.5M) and 1ul glycoblue co-precipitant. Store at -20oC overnight.
SECTION: Lithium Chloride Precipitation
35. Centrifuge at max speed for 25 min, 4oC. You should see large, gelatinous pellet form at bottom of tube.
SECTION: Lithium Chloride Precipitation
36. Wash 2X with 750µl ice-cold 75% ethanol. Vortex tubes ~30s to completely wash tube and pellet. Centrifuge max speed for 5 minutes, 4oC.
SECTION: Lithium Chloride Precipitation
37. Remove all ethanol from pellet (remove supernatant, quick spin, remove residual ethanol with aspirator). Air dry pellet for ~5 min if a lot of residual ethanol remains.
SECTION: Lithium Chloride Precipitation
38. Resuspend pellet in 30 µL 50oC nuclease-free water.  Make sure pellet is completely resuspended before moving forward!