AssemblyLink
The group performing the AssemblyLink protocol (Cantata Bio) was led by Lily Shue (Cantata, US). Group participants were Maria Gyftea (SciLifeLab, SE), Eunkyong Choi (SciLifeLab, SE), Laurie Bertrand (Genoscope, FR), Carmela Gissi (UniBari, IT), and Maria Laura Aguilera Gil (CNAG, ES)
- This content is currently in post-production and access is limited to participants
- If you require access, please contact the course administrators
Tissue preparation
Sample preparation was performed by each participant according to how they usually disrupt tissues.
Herring tissue was disrupted in a mortar and pestle on wet ice with addition of liquid nitrogen. Runtime: 8.5 minutes.
Reindeer heart tissue was disrupted in a mortar and pestle laying in liquid nitrogen. Runtime: 10 minutes.
Sample preparation: Reindeer heart
Reindeer spleen tissue was disrupted in a mortar and pestle laying in liquid nitrogen. Runtime: 7.5 minutes.
Sample preparation: Reindeer Spleen
The small krill sample was disrupted in a mortar and pestle laying in liquid nitrogen. Runtime: 7 minutes.
The moose sample was preserved in ethanol. It was washed in PBS and disrupted in in a mortar and pestle laying in liquid nitrogen. Runtime: 7 minutes.
Lysate preparation
The AssemblyLink protocol was used when crosslinking and creating a lysate from the samples. Maria Gyftea demonstrates this protocol on the krill sample, including discussion with Lily. Runtime: 17 minutes.
Lysate QC
The quality control protocol is performed on all samples in order to check if enough lysate has been created for the downstream procedures, and to determine its concentration and quality. The lysates are found to have very low yields. Runtime: 15.5 minutes.
The lysates are run on the bioanalyzer to determine the quality of the lysate, and found to be underdigested. Runtime: 4 minutes.
Proximity Ligations
While Lily repeats the AssemblyLink Sample preparation with higher input, the herring and reindeer spleen samples are chosen to proceed with the protocol. In the first step, chromatin capture is performed. Runtime: 4.5 minutes.
In the next step, three ligation reactions and the crosslinking reversal reaction are performed. Runtime: 11 minutes.
Finally, the DNA is purified from the sample lysate. Runtime: 7 minutes.
Library Preparation
In the final step of the protocol performed during the course, library preparation is performed on the herring and reindeer spleen samples from the first round. The protocol is performed up until the Index PCR stage, at which point the samples were stored for further analysis. Runtime: 8.5 minutes.
Troubleshooting and additional attempts
The results of increasing input are not promising, as the samples are still underdigested. The AssemblyLink group discusses possible additions and controls. Runtime: 8 minutes.
A third attempt is performed on the herring and reindeer spleen tissues with the inclusion of the cell isolation module. Additional controls and discussion is included, and we recommend watching the first sample preparation video first! Runtime: 24 minutes.