Please note that the following protocols are patented or patent pending. We recommend to consult us for advises of optimized processing of your specific samples.
For RNC-seq of cultured animal cells (anchorage-dependent cell)
Cell number needed:
Depending on the cell line. As a reference, 5 million cells is sufficient for cancerous cell lines.
100x CHX solution: Dissolve 50mg cycloheximide (CHX) in 5ml RNase-free water. This solution can be stored in -20°C.
1. Add 1% volume of 100x CHX solution into the cell culture medium.
2. Incubate the cells in the previous culturing environment for another 15min.
3. Remove the culture medium and dissociate cells gently but as quick as possible. If you cannot dissociate cells easily from the plate or flask, proceed to the protocol (B) below.
4. Transfer the dissociated cells into a 1.5ml RNase-free tube and put on ice.
5. Rinse the cells once with pre-chilled PBS buffer. Remove the PBS buffer after rinsing.
6. Shock-freeze the tube into liquid nitrogen.
3. Remove the culture medium and rinse the cells gently using pre-chilled PBS buffer. Then remove the PBS buffer. Put the culture plate or flask on ice.
4. Add pre-chilled PBS buffer. All cells must be immersed in PBS buffer.
5. Shock-freeze the plate or flask in liquid nitrogen. You need to carefully keep the balance of the plate or flask to ensure that the cells are immersed in PBS buffer, and no liquid nitrogen comes into the plate or flask.
The cell sample can be preserved in -80°C for at least one month. Transport with dry ice.
For RNC-seq of animal tissue
We have succeeded RNC-seq for at least 12 major organs in mouse. However, animal tissue samples are highly risky. It is extremely difficult to extract enough amount of RNC from some special tissue samples. The following pre-treatment protocol works for at least half of the tested tissues.
It’s highly dependent on the tissue. 100mg or 0.5mm3 would be a nice choice for the first trial.
Protection solution: DPBS solution + 0.3mg/ml cycloheximide + 50mM MgCl2. pH=7.0~7.2. This solution should be stored at 0~4°C. Prolonged storage is not recommended.
1. Cut quickly the tissues into <0.5mm3 slices. If you cannot do it quickly, you have a second chance to do it in the next step.
2. Incubate and rinse each slice in 1-2ml protection solution for 2-3min on ice. If you need to cut the tissues into smaller pieces, do it quickly in the protection solution. Then remove the protection solution.
3. Transfer the tissue sample into a new 1.5ml tube. Incubate and rinse each slice in 1-2ml protection solution for 3-5min on ice.
4. Remove the protection solution. To avoid loss, DO NOT use filter paper to dry tissues thoroughly.
5. Transfer the tissue sample into a new RNase-free 1.5ml tube. Immediately shock-freeze the sample in liquid nitrogen until it’s completely frozen.
The tissue sample can be preserved in -80°C for 6 months. Transport with dry ice.
For mRNA sequencing
Please dissolve the cells or tissues in sufficient amount of TRIzol reagent. Refer to the TRIzol manual for more information. We recommend >3 million cells or 100mg tissues for RNA sequencing.
0°C or lower temperature is needed for international transport.