Tendon Seed Network Protocols
This protocol is used the the Tendon Seed Network to extract nuclei from small tissue biopsies of Tendon tissue. For larger pieces opf tissue we use the GentleMACS protocol.
This protocol is an iteration of various nuclei extraction protocols (Martelotto, Humphreys, Macosko) developed specifically for tendon single-cell RNA-seq experiments, using drop-seq based techniques. The protocol can be used to isolate nuclei from both small (less than 3mmx3mm) pieces of tissue. The protocol has an optional (but recommended) FACS based isolation step if the sample contains lots of debris that can make nuclei counting difficult and sample blockages in the microfluidics more likely. However, this leads to significantly increased loss of nuclei so you may not have enough to perform your single-nuclei experiments at the end of the protocol.
The protocol used has been developed iteratively using healthy snap-frozen hamstring tendon but should be amenable for any tendon.
The protocol is used routinely within the tendon seed network for single nuclei experiments using drop-seq methods.