title: SMRT sequencing of Eimeria necatrix: second-generation merozoites
design: After first-strand synthesis, large-scale double-strand cDNA was generated by polymerase chain reaction (PCR) amplification using Phusion DNA polymerase (NEB, Beverly, MA, USA). This procedure allows for detection of full-length transcripts up to 4 kb without size selection. To increase the yield of > 4 kb transcripts, size selection was performed using the BluePippin Size Selection System (Sage Science, Beverly, MA, USA) protocol described by PacBio (PN 100-092-800-03). Subsequently, both ends of the cDNA were ligated with SMARTer adaptors (PacBio) and SMRTbell (PacBio) to produce circular single DNA molecules, which were sequenced using a PacBio RS sequencing instrument.