GSM8950898: P. australis, EU, rep2; Phragmites australis; RNA-Seq
The Rhizome tip was cut into 1-2 mm strips and added to tubes containing 10ml enzyme solution consisting of 2% cellulase R10, 0.8% macerozyme R-10, 0.5% pectinase Y-23, 0.1% BSA, 20mM MES,20mM KCl,10mMCaCL2 and 0.55M mannitol with shaking at 60rpm for 1 hour at 28℃. Cells were then filtered with a 40 um cell strainer. Cell activity was detected by trypan blue staining and cell concentration was measured using a hemocytometer and a light microscope. Lastly, protoplasts were resuspended in 8% (w/v) mannitol solution in preparation for loading onto the chromium controller of the 10xGenomics platform. Cells were loaded onto the 10X Chromium Single Cell Platform (10X Genomics) at a concentration of 700-1200cells/ul (Single Cell 3′ library and Gel Bead Kit v.3) as described in the manufacturer's protocol. Generation of gel beads in emulsion (GEMs), barcoding, GEM-RT clean-up, complementary DNA amplification and library construction were all performed as per the manufacturer's protocol.