Raw reads: Nanopore-Spironucleus_SBKI-1
unspecified
S. barkhanus trophozoites (200 mL of culture, ~2×108 cells) were harvested by centrifugation for 10 min, 500×g, 4°C. The cells were washed with 100 mL of PBS and re-pelleted as above. The washed cells were lysed in 2 mL lysis buffer (1% SDS, 50 mM EDTA, 30 mM DTT and 800 µg Proteinase K (Thermo-Scientific, EO0491)) at 56 °C for 2 hrs. Nucleic acid was extracted using two rounds of Phenol-Chloroform-Isoamylalcohol (25:24:1) and one round of Chloroform-Isoamylalcohol (24:1) separation followed by precipitation using 0.7× volumes of isopropanol. The isolated nucleic acid was treated with 500 µg of RNase A for 30 min at 37 °C. The DNA isolated using a Qiagen Genomic Tip G/20 according to the manufacturer's instructions. High molecular weight genomic DNA was Genomic DNA was selected using the Short Read Eliminator (SRE) (Circulomics, SS-100-101-01). DNA concentrations were determined using the Qubit dsDNA BR kit using a Qubit 2.0 (Invitrogen). The size distribution of the DNA was determined using agarose gel electrophoresis. DNA purity was determined by NanoDrop 1000 Spectrophotometer v3.8.1 (Thermo Scientific). Library preparation was accomplished using 1500 ng of SRE selected genomic DNA using the Oxford Nanopore ligation sequencing gDNA kit (SQK-LSK112), version GDE_9141_v112_revE_01Dec2021 and sequenced on an R10.4 flow cell (FLO-MIN112) using MinION Mk1C running MinKNOW v21.11.6 software. The raw nanopore data were basecalled using Dorado version 0.7.1 in super-accuracy mode using the dna_r10.4.1_e8.2_260bps_sup@v4.1.0 model.