RNA-seq of Tetrahymena: conjugation 10h
Tetrahymena wild-type strains (SB210 and CU428) were obtained from the Tetrahymena Stock Center (http://tetrahymena.vet.cornell.edu). Cells were grown in SPP medium at 30C. For conjugation, starved SB210 and CU428 cells were resuspended in 10 mM Tris (pH 7.5) at 2  105 cells/ml, mixed in equal volumes, and samples were collected at 4h, 5h, 6h, 8h, 10h after mixing. Total RNA was collected using the RNeasy Plus Mini kit (Qiagen, 74134). The quality and concentration of RNA samples were analyzed by 1% agarose gel electrophoresis and Qubit3.0 Fluorometer (Thermo Fisher Scientific). Strand-specific RNA sequencing libraries and Illumina sequencing libraries of SB210 were constructed according to manufacturer-recommended protocols and sequenced by Novogene Co. Ltd (Beijing, China). For ATAC-seq libraries, transposase Tn5 from the Nextera DNA Library Preparation Kit (#FC-121-1030, Illumina, USA) was employed to treat 105 macronuclei for 1h at 37C. The DNA from the samples was subsequently recovered using the MinElute Recovery Kit (#28004, Qiagen, Germany). Amplification and library construction of the sample DNA were performed for 13 PCR cycles, with library adapter primers sourced from the Nextera XT Index Kit (#FC-121-1011, Illumina, USA). The DNA from the constructed library was recovered once more using the MinElute Recovery Kit (#28008, Qiagen, Germany).