title: re-sequencing of TXR1 knockout Tetrahymena thermophila growth cells
design: Tetrahymena thermophila WT427and ?TRR1cells were used to extract the total DNA. Cells were cultured in 1x SPP medium (Asai, 2000), and cell density was counted using Beckman Count Coulter Z1 (Beckman Coulter, Brea, CA, USA). Cells were collected at ~2x105 cells/ml, lysed with 2X urea buffer (42% urea, 0.35 M NaCl, 0.01 M Tris pH 7.4, 0.01 M EDTA, 1% SDS), and total DNA was extracted using the phenol-chloroform method (Sambrook and Russell, 2001). DNA integrity was verified using gel electrophoresis with 1.2% Agarose. DNA concentration was measured using e-Spect ES-2 spectrophotometer (MALCOM, Japan).      Libraries were constructed using the TruSeq DNA Sample Preparation Kits v2 (Illumina, San Diego, CA, USA, FC-121-2001).DNA was randomly sheared into small fragments (~ 210 bp) using Bioruptor(Diagenode SA., Liege, Belgium), according to the manufacturer’s recommendations. The fragment overhangs were repaired to blunt ends using T4 DNA Polymerase,T4 Polynucleotide Kinase, and DNA Polymerase I Klenow fragment(Illumina). Illumina adaptors (Illumina) were then ligated to the DNA fragment using DNA ligase(Illumina). Finally, polymerase chain reaction (PCR) was performed using DNA Phusion Polymerase (Illumina)to amplify DNA and enrich those DNA fragments that ligated with adapters on both ends. Cluster formation, primer hybridization and pair-end sequencing were performed using proprietary reagents and IlluminaHiSeq 2000 (Illumina) according to manufacturer-recommended protocols (https://icom.illumina.com/).