Notes

Bacteria were washed away from choanoflagellate cells through two rounds of centrifugation and resuspension in artificial seawater. To count the cell density, cells were diluted fold in l of ASW, and fixed with L of paraformaldehyde.Cells were counted on a hemocytometer, and the remaining cells were diluted to a final concentration of choanoflagellate cellsmL.mL aliquots and plated in six well plates prior to treatment.After treatment, cells were transferred to a mL conical and pelleted by centrifugation at xg for min, flash frozen with liquid nitrogen, and stored at C.Double the amount of lysis buffer was used to increase RNA yield and decrease degradation, and RNA was eluted in minimal volumes in each of the two elution steps. To remove contaminating bacterial RNA, samples were first poly A selected using oligo dT attached magnetic beads.Following purification, the mRNA was fragmented at C for min, and cleaved RNA fragments were synthesized into cDNA.Duplicates were marked using picard tools, and <a href="https://www.ncbi.nlm.nih.gov/pubmed/22217876">AMG 837</a> differential expression analysis was performed using edgeR. Statistical cutoffs of FDR were used to identify significant differentially expressed genes.GO enrichment analysis of differentially expressed genes was performed using DAVID. Membranes were incubated with primary antibodies diluted in PBST overnight at C and washed extensively in PBST., and serum was affinity purified against the peptide to reduce cross reactivity and validated using immunoblotting.After the cells were allowed to settle on the coverslip for min, l of the cell solution was gently removed from the side of the dish.Next, l cold paraformaldehyde diluted in X PBS was added, and the fixative mixture was incubated for min at room temperature.After fixation, the coverslip was gently washed three times with l X PBS.Cells were permeabilized by incubating in permeabilization buffer for min.After removing permeabilization buffer, the coverslip was incubated in primary antibody for hour at room temperature, and then washed three times in X PBS.The coverslip was then incubated with secondary antibody for hour at room temperature, and then washed twice in X PBS.The mounting media cured overnight before visualization.The culture flasks were combined and vigorously shaken for s, and then transferred to ml conical tubes and spun for min at g and C.The supernatant was removed with a serological pipette, and residual media were removed with a fine tip <a href="https://www.targetmol.com/compound/AMG%20837%20calcium%20hydrate">buy AMG 837</a> transfer pipette.After the supernatant was removed, the cells were resuspended in a total volume of l of ASW.To count the cell density, cells were diluted fold in l of ASW, and fixed with l of paraformaldehyde.Cells were counted on a hemocytometer, and the remaining cells were diluted to a final concentration of choanoflagellate cellsml.The resuspended cells were divided into l aliquots with cells per aliquot to immediately prime cells in the next step.; mM lithium citrate; mM l cysteine; PEG; and M papain to remove the extracellular material coating the cell.The l aliquots, which contained cells, were centrifuged for min at g.The supernatant was removed, and cells were resuspended in l of priming buffer and then incubated for min at room temperature.Priming was quenched by adding l of mgml bovine serum albumin fraction V and then centrifuged for min at gand C with the centrifuge brake set to a soft setting.