RNAseq from A. castellanii Rep1 time 1h  in encysting medium
A. castellanii trophozoites, were seeded into seven 25 cm2 culture flasks at a density of 5  105 cells in 10 mL of PYG medium and incubated at 30 C overnight. Afterward, for growth condition, PYG medium was replaced by 10 mL of fresh PYG medium (P1, P4, P8/Time, 1h, 4h, 8h).  For encysting condition, cells were rinsed twice with 10 ml of Pages Amoeba Saline (PAS) buffer (0.1% sodium citrate dihydrate, 0.4 mM CaCl2, 4 mM MgSO4, 0.05 mM Fe(NH4)2(SO4)2 6H2O, 2.5 mM K2HPO3, 2.5 mM NaH2PO3, pH 6.5) and then 10 mL of encysting buffer (E1,E4,E8/Time1h, 4h, 8h) (0.1 M KCl, 0.4 mM CaCl2, 1 mM NaHCO3, 8 mM MgSO4, and 20 mM 2-amino-2-methyl-1,3-propanediol, pH 8.8) were added to the culture flasks. Cells were incubated at 30C for 1, 4 or 8h. Following incubation, cells were rinsed twice with PAS and total RNA was extracted with a RNeasy Mini Kit (QIAGEN, Germany). As control condition, one flask did not receive any fresh medium after overnight incubation (P0/0h), cells were rinsed twice with PAS right away and total RNA extraction was carried out using a RNeasy Mini Kit (QIAGEN, Germany). The sequencing was done by GENEWIZ genomics service company (Leipzig, Germany), using the paired-end Illumina HiSeq libraries (2150 bp reads).
AC_E1_Rep_1