Chesler5 project protocol

Intestinal microbiota in 10 inbred strains of mice   (2012)

Chesler EJ, Bubier JA, Podar M
With: Campbell JH, Foster CM, Vishnivetskaya T, Campbell AG, Yang ZK, Wymore A, Palumbo AV




Chesler5 Protocol

Project protocol - Contents

Workflow and sampling

Step
Procedure
Data collected *
1
 Mice euthanized
-
2
 Jejunum and cecum dissected and flushed
-
3 Microbial genomic DNA extracted -
4 Small subunit (SSU) rRNA sequenced (V4 region) -
5 Sequence analysis of operational taxonomic units (OTU) Identification of OTUs and relative abundance determined

*Supplementary data are available for this project. See Chesler5

Equipment and supplies

  • MoBio vortex adapter
  • Shaker
  • Phase gel lock tubes (Qiagen, Valencia CA)
  • Agilent Bioanalyzer (Santa Clara CA)
  • 454-FLX Sequencer (Roche, Indianapolis IN)
  • Mothur software (V1.11.0)

Reagents and solutions

  • Phosphate buffered saline (PBS)
  • Silica/zirconia beads (0.1 mm; BioSpec Products, Bartlesville OK)
  • Phenol:chloroform:isoamyl alcohol (25:24:1)
  • Sodium dodecyl sulfate (SDS)
  • Tris, pH 8.0
  • NaCl
  • EDTA
  • Ammonium acetate
  • Sodium acetate
  • Isopropanol
  • Ethanol
  • TE buffer
  • RNAse A
  • AccuPrime Pfx reaction mix (Invitrogen)
  • Barcoded primers (Integrated DNA Technologies, Coralville IA)
  • Agencourt AMPure reagents (Beckman Coulter, Danvers MA)
  • DNA 1000 reagents (Santa Clara CA)

Procedure: Jejunum/cecum dissection and preparation of contents

    1. Mice are euthanized by cervical dislocation at the same time each day.
    2. A 12-cm long segment of jejunum starting 5 cm distally from the ligament of Trietz is dissected (cecum is included).
    3. Cecum contents are extruded manually and snap frozen in liquid nitrogen and stored at -80°C.

Procedure: Extraction of microbial genomic DNA

    1. Microbial genomic DNA is extracted from mouse cecum contents using a protocol based on Ley et al (2008) where approximately 100 mg of cecum contents is added to a 2-mL screw-caped tube containing 1 g of silica/zirconia beads, 500 µL of phenol:chloroform:isoamyl alcohol, and 210 µL of 20% SDS. Headspace is filled with cold DNA extraction buffer (200 mM Tris pH 8.0, 200 mM NaCl, 20 mM EDTA).
    2. Bead tubes are attached to a MoBio vortex adapter and shaken horizontally at high speed for 10 min.
    3. The aqueous phase is washed three times with phenol:chloroform:isoamyl alcohol in phase gel lock tubes.
    4. Nucleic acids are precipitated with 1 vol ammonium acetate (7.5 M), 2 vol isopropanol and incubated at -20°C for at least 1 h.
    5. Precipiated nucleic acids are concentrated by centrifugation at 15,000 g for 15 min then dissolved in TE buffer.
    6. RNase A (100 U) digestion is performed for 30 min at 37°C.
    7. Genomic DNA is precipitated with 0.1 vol sodium acetate (3 M, pH 5.5) and 3 vol ethanol and incubated at -20°C for at least 1 h.
    8. DNA is concentrated by centrigugation at 15,000 g for 15 min, pellets are washed twice with 70% ethanol, air dried and dissolved in PCR-grade water (mock extractions without cecum contents are used as negative controls).

Procedure: Preparation and pyrosequencing of SSU rRNA gene amplicon libraries

    1. V4 amplicons (V1-2 amplicons are reported in Campbell et al (2012) but are not included in the Chesler5 dataset) are generated in 50 µL reactions composed of 1X AccuPrime Pfx reaction mix, 300 nM forward primer, 300 nM reverse primer mix, 1.5 U AccuPrime Pfx polymerase and 100 ng of genomic DNA. For more information, see Campbell et al (2012).
    2. Thermal profiles consist of a denaturation step at 95°C for 2 min, followed by 27 amplification cycles of 95°C for 15 s, 55°C for 30 s, and 68°C for 45 s, followed by a final extension at 68°C for 3 min.
    3. Amplicons are visualized on agarose gels for quality and subsequently purified using Agencourt AMPure reagents.
    4. A final check of amplicon quality and quantity is performed on an Agilent Bioanalyzer using DNA 1000 reagents.
    5. Sequencing is performed on a 454-FLX instrument following manufacturer's instructions.
    6. Sequences are extracted from raw FASTA files using the Ribosomal Database Project (RDP) Pipeline Initial Process.
    7. Mothur is used to further screen sequences for each SSU rRNA gene region. See Campbell et al (2012) for more details.
    8. Representatives of each OTU are collected at genetic distances of 0.03 using Mothur.
    9. RDP Naive Bayesian rRNA Classifier is used for taxonomic analysis of all sequences.

Data collected by investigator

  • OTU-based sequence analysis and relative abudance

*Supplementary data are available for this project. See Chesler5