Dneasy blood mini kit

Clonal cultures of two Cryptomonas species were established from single cells isolated manually from natural habitats by glass pipetting: C. curvata KR (FBCC300012D), from Cheongyang, Korea (36° 30′ N, 126° 47′ E), and C. paramecium KR from freshwater, Daejeon, Korea (36° 21′ 57″ N, 127° 20′ 20″ E). The strains have been deposited in, and are available from, the Freshwater Bioresources Culture Collection at the Nakdong-gang National Institute of Biological Resources and the Protist Culture Collection, Department of Biology, Chungnam National University, Korea. The two cultures were grown in AF-6 medium [42 ] with distilled water and were maintained at 20°C under a 14:10 light:dark cycle with 30 μmol photons m⁻² s⁻¹ from cool white fluorescent tubes. Cultivation of C. curvata CCAP979/52 and Cryptomonas sp. CCAC1634B was carried out as described [16 (link)].
Genomic DNAs were extracted from C. paramecium KR and C. curvata KR (FBCC300012D) using the QIAGEN DNEasy Blood Mini Kit (QIAGEN, Valencia, CA, USA) following the manufacturer’s instructions. DNA extractions for C. curvata CCAP979/52 and Cryptomonas sp. CCAC1634B were done using a standard SDS-phenol/chloroform extraction method. For C. curvata CCAP979/52, organelle DNA-enriched fractions (i.e., plastid, mitochondrion, and nucleomorph) were purified as described previously [11 (link)].

A culture derived from a single-cell isolate of Teleaulax amphioxeia collected from Gomso Bay, Korea (35° 40’ N, 126° 40’ E), which was established in a previous study [56 ], was selected for genome sequencing. DNA was extracted from the cultivated sample using a QIAGEN DNEasy Blood Mini Kit (QIAGEN, Valencia, CA, USA), following the manufacturer’s instructions. A sequencing library was prepared using an Ion Xpress Plus gDNA Fragment Library Preparation Kit and an Ion OneTouch 200 Template Kit v2 DL (Life Technologies, San Francisco, CA, USA) according to the manufacturer’s protocol and sequenced with an Ion Torrent Personal Genome Machine (PGM) at the Yoon laboratory at Sungkyunkwan University (Suwon, Korea) using an Ion PGM Sequencing 200 Kit v2 (Life Technologies, San Francisco, CA, USA).

Qiagen DNeasy blood mini kit (Qiagen) and NucliSENS easyMag instrument (BioMerieux) were used to extract DNA from PBMC and plasma, respectively, in accordance to the manufacturer’s instruction. Part of the DNA samples were transferred to The University of Birmingham and the HLA laboratory in Queen Mary Hospital, The University of Hong Kong for HLA typing. The HLA types of all subjects recruited in ELISPOT assay were documented (Supplementary Table 3). The rest of the DNA was used to measure the EBV loads by qPCR with ABI PRISM 7900 sequence detector (Applied Biosystems, United States). EBV loads in PBMC were determined by the amplification of viral DNA polymerase (Pol, BALF5) sequence. Human β2 microglobulin sequence was detected as an internal control. Plasma EBV loads were quantified by expression of the BamH1W repeats in the EBV genome.