Blepharisma stoltei developmental sRNA-seq time course

Non-gamone treated Blepharisma stoltei strain ATCC 30299 (A1) and HT-IV cells (H1) were collected across a conjugation/developmental time course, starting at 3 hours before mixing the strains. The ATCC 30299 cells were then treated with synthetic Gamone 2 and the HT-IV cells were treated with cell-free fluid with a Gamone 1 activity of 512 U/ml for two hours.After three hours ATCC 30299 cells only and HT-IV samples only were collected separately for RNA extraction. Two cultures were mixed together to initiate heterotypic pairing, constituting the experiment's 0 hour time point. Cell samples were collected for RNA isolation at 2 h, 6 h, 14 h, 18 h, 22 h, 26 h, 30 h and 38 h. All samples were harvested for RNA-extraction using Trizol. The total RNA obtained from Trizol extraction was then separated into a small RNA fraction less than 200 nt and a fraction with RNA fragments greater 200 nt using the Zymo RNA Clean and Concentrator-5 kit according to the manufacturer's instructions. The small RNA fraction was further size selected on a PAGE gel in the 18-30 nt size range, and used to prepare a standard BGI ssCir DNA sRNA-seq library.