Sample preparation is a crucial step in translatomics research. Here, Creative Biolabs provides some guidelines related to collection and transport of samples to ensure a smooth project phase.
Sample Handling Precautions
During the sampling process, the sampling time, location and operation process should be consistent. Otherwise, the reproducibility and reliability of the experimental results may be compromised.
Sample Types
We generally accept the following sample types for ribosome profiling and polysome profiling analysis:
Please consult with us for other sample types.
Cell Samples
Regardless of adherent cells or suspension cells, a minimum of 1X107 cells are required.
(1) Ensure that adherent cells are in good growth status. Seed cells prior to desired processing time.
(2) When the cell confluency reaches about 70%, add cycloheximide (CHX) to the culture medium to block translation, and incubate cells at 37°C for 15 min.
(3) After digestion with trypsin, terminate the digestion with complete culture medium. Transfer the cell suspension to a centrifuge tube, centrifuge, discard the supernatant, add PBS containing CHX, wash 3 times, centrifuge to remove the supernatant, quickly freeze in liquid nitrogen and store at -80°C.
(1) Seed cells at 48 hours or before the required processing time.
(2) When the cells reach 70-90% confluence, process as adherent cells, add CHX and incubate.
(3) After collecting the cells by centrifugation, same as adherent cells, add pre-cooled PBS and wash, remove the supernatant, freeze in liquid nitrogen and store at -80°C.
Notice:
No liquid residue.
Cell handling should be gentle.
Bacterial Samples
(1) Inoculate strain into sterile medium for overnight culture.
(2) Culture the cells to the growth exponential phase, add chloramphenicol to continue the culture.
(3) Pour the bacterial solution into two pre-cooled centrifuge tubes and invert.
(4) Centrifuge at 12,000g at 4°C for 6 minutes, and discard the supernatant.
(5) Add pre-cooled buffer to resuspend and wash.
(6) Centrifuge at 3000g at 4°C for 5 minutes, and discard the supernatant.
(7) The samples were rapidly frozen in liquid nitrogen and stored at -80°C.
Yeast Samples
(1) Yeast was cultured under standard conditions. Add CHX, and continue to incubate.
(2) Samples were collected by centrifugation at 12,000g for 10 min at 4°C.
(3) Centrifuged cells were quick-frozen in liquid nitrogen and stored at -80°C.
Tissue Samples
(1) Prepare surgical tools, ensure an RNase-free operating environment.
(2) Follow the standard laboratory procedure to quickly euthanize the animal and collect the target tissue.
(3) Wash the tissue with RNase-free PBS to remove excess connective tissue and blood.
(4) Rapidly freeze the tissue in liquid nitrogen, grind it into powder, and transfer it to a cryovial.
(5) Store at -80°C, transport on dry ice.
Storage of Samples
After sampling, simply handle and label, and store at -80°C to avoid sample degradation.
Submission Guidelines
Proper sample transport is the guarantee for smooth analysis. Please note the following instructions:
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Please ship samples on dry ice.
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Provide sufficient dry ice based on the quality of the insulated shipping container and sample.
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Ensure that the packaging is firmly sealed and labeled correctly.
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Please inform us in time before sending the sample, including the sample submission form and tracking number, so that we can register and process the sample after the arrival of the samples.
Please contact us for more details.
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