title: Subclone A2E10
organism: Plasmodium falciparum
description: Protocols: The 7G8 subclones were grown at a hematocrit of 5% in human O+ erythrocytes by standard procedures (Trager W & Jensen J 1976. Human malaria parasites in continuous culture. Science 193:673-675. doi: 10.1126/science.781840). Cell cultures were regularly synchronized with 5% sorbitol (Lambros C & Vanderberg JP 1979. Synchronization of the erythrocytic stages of Plasmodium falciparum in culture. J Parasitol 65:418-420. doi: 10.2307/3280287), with only ring stage parasites surviving this treatment. Samples for gDNA-seq were obtained by centrifugation of 150 ml of a P. falciparum cell culture at 800 x g, and red blood cell pellets were lysed in a cell lysis solution containing proteinase K. Genomic DNA was purified according to the manufacturer's instructions \cell samples\ followed by \complete removal of RNA\ with additional RNase I treatment of the MasterPure™ Complete DNA Purification Kit (Lucigen). This included gentle lysis of red blood cells, removal of cell debris and proteins by precipitation, and RNase I treatment in solution, using no phenol, chloroform, or guanidinium salts to avoid interference with downstream NGS sequencing. The gDNA samples were checked for concentration, sample integrity, and purity. Concentration was detected with a fluorometer using the Qubit™ dsDNA BR Assay Kit (ThermoFischer). Sample integrity and purity were detected by agarose gel electrophoresis. Library construction of the gDNA was performed by BGI Genomics Co, Hong Kong. A PCR-free library protocol could be used for the A2E10 and A2G2 subclone samples; the A1G9 subclone sample required a PCR amplification step after adaptor ligation. Per sample, 1 μg of genomic DNA was randomly fragmented by Covaris. The fragmented genome, with an average size of 300-400 bp, was selected with magnetic beads and quantified with a Qubit fluorometer. The fragments were subjected to end repair and then 3'adenylated. Adapters were ligated to the ends of these 3'adenylated fragments. The double-stranded products were heat denatured and circularized through the splint oligo sequence. The single-stranded circular DNA (ssCir DNA) was formatted as the final library. The library was qualified using the Agilent Technologies 2100 Bioanalyzer.