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SNOWGLOBE_HYPOXIC_TIME_COURSE_PROTOCOLX - BROAD INSTITUTE

By Kenneth Gonzales,2014-07-04 08:39
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PREPARE CULTURE FOR HYPOXIC TIME COURSE ... EXPAND CULTURE INTO 100 ML 7H9 IN A 500 ML ROLLER BOTTLE. INCUBATE ... FILL EACH STERILE SPINNER FLASK WITH 500 ML CULTURE.

SnowGlobe (SG) Protocol

    Prep work: Label tubes

     Prepare sufficient media:

     3L Sauton’s with no detergent (SnoT)

     7H9 for inoculating culture (~1L)

     Sterilize and prep spinner flasks

Expected output of this experiment:

    - Cell pellets and supernatant from a hypoxic and reaeration time course

    - An excel spreadsheet of the entire experiment

    o A sheet logging dates and times of samples

    o A description of the clumpy culture if anything unusual is visible

    The first challenge to setting up this type of experiment is preparing sufficient culture necessary to get almost 3L of snowglobe adapted log phase culture to start the experiment: 4 vessels with 500 mL/vessel plus the 600 mL for the T0 samples plus 60 mL for sterility checks. To get this volume of biomass we start in 7H9, grow them to half of the desired starting OD, and then transfer to SnoT media for two days to let them adapt during approximately one doubling.

Prepare culture for hypoxic time course

    1. Inoculate 9 mL 7H9 with 1 mL frozen stock H37Rv (from CSU via Stanford) in a 50 mL

    PP tube. Incubate in a rotator at 37?C to an OD >1. Use this as a fridge stock. 600

    2. Expand culture into 100 mL 7H9 in a 500 mL roller bottle. Incubate in a roller at 37?C to

    an OD of 1.0. 600

    3. Expand culture again into 4 X 200 mL 7H9 in four 1L roller bottles. Incubate in a roller

    at 37?C to an OD of 0.4-0.5. 600

    4. Pellet cells at 5K g for 5 minutes in 16 X 50 mL aliquots. Wash pellets 1X in 10 mL PBS.

    Resuspend four pellets in 50 mL SnoT media, transfer to a 2L bottle with 350 mL SnoT

    media (400 mL total). Repeat for each set of four pellets. OD, were it measurable,

    should be at 0.2

    5. Incubate for 2 days at 37?C in rolling culture, allowing ~one doubling.

    6. Dilute with an equal volume of SnoT media to get 3.2L of culture at ~0.2 starting OD.

    7. Set aside 15 mL of the SnoT media for metabolomics. Store at -80C

Prepare spinner flasks and take T0.

    1. Fill each sterile spinner flask with 500 mL culture.

    2. Attach to 0.2% O gas line and begin hypoxia. 2

    3. Pellet remaining culture for T0 and sterility test samples (at least 20 cell pellets).

    a. Save supernatant from each set of pellets

    b. Resuspend 4 pellets in Trizol for RNA

    c. Re-suspend 8 pellets in 1.5 ml 6M guanidinium thiocyanate in prelabeled 2 ml

    screw cap tubes.

    d. Store the remaining pellets and the treated samples at -80?C until the end of the

    experiment

    At each hypoxic and reaeration time-point:

    Note: From 500 mL of hypoxic culture a maximum of 20 x 25 mL aliquots can be taken.

    When sampling for all cores that sets a limit of

    1. One flask at a time;

    a. Detach from the gas line and bring in to the BSC

    b. Take 5x30 mL samples (150 mL total) and place on ice.

    c. Return flask to the stir plate and reattach gas lines

    2. Pellet samples

    a. Resuspend 2 pellets from each set in GTC, as above

    b. Resuspend 1 pellet from each set in Trizol

    c. Save the other 2 pellets for C:M treatment

    Sterilization and sample processing

    1. Prepare two buckets of dry ice. Remove entire collection of samples from the hypoxic

    time course from the -80?C freezer onto dry ice and bring into BL3 suite. 2. Separate and sort tubes into replicate sets (one tube from each timepoint). 3. For the proteomics pellet sets (proteomics will receive 2 processed pellets and 50 mL

    supernatant for each time point from each flask):

    a. Incubate GTC treated pellets overnight in a rotator at 37?C.

    b. Transfer sterilized pellets to a -80?C freezer until shipping to PPD.

    c. Check sterility of GTC treated cells by plating a control cell pellet.

    d. Wait until colonies would normally appear- a minimum of 21 days.

    e. Once samples are confirmed sterile, ship to PPD on dry ice as non-infectious

    material along with the supernatant samples.

    4. For the lipidomic and metabolomic pellet sets:

    a. Re-suspend pellets (which must be 0.5 ml in volume or less) in 10 mL C:M (2:1,

    V:V), cap tightly, and vortex for one minute to distribute solvent. Let it sit for 1

    hr at room temperature to sterilize.

    b. Place tube on a rotating platform, and extract for an additional 1 hr at room

    temperature. Treat outside of tube with chemical disinfectant and remove from

    BSL-3.

    c. Transfer to a glass tube and evaporate to dryness under nitrogen. Store samples at

    4?C until ready to ship.

    d. Check sterility of C:M treated cells by plating using a control cell pellet.

    e. Wait until colonies would normally appear- a minimum of 21 days.

    f. Once samples are confirmed sterile, ship to Metabolon and BWCH on dry ice as

    non-infectious material.

    5. For the transcriptomic pellet set:

    a. Standard RNA prep protocol.

    This experiment will generate 4 time points from four replicates. Therefore we should have in the freezer at the end:

    16 C:M treated pellets for BWH (lipidomics)

    16 C:M treated pellets, 16 supernatant samples, and 1 media sample for Metabolon (metabolomics)

    2 sets of 16 GTC treated pellets and 16 supernatant samples for Caprion (proteomics) 16 RNA samples for transcriptomics.

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