title: GSM6047392: ME49_TgCycP2_Tz_-aux_rep1; Toxoplasma gondii; RNA-Seq
library_construction_protocol: Total RNA was extracted with TRIzol according to the manufacturer's instructions (Invitrogen). The RNA integrity was verifed using Agilent High Sensitivity D1000 ScreenTape in an Agilent 2200 TapeStation System (Agilent Technologies). One µg of total RNA was treated with 1 U of DNase I (Amplication Grade, Invitrogen) to remove the residual genomic DNA, and the DNase-treated RNA was used as an input for the construction of RNA-seq library using IlluminaTruSeq® Stranded mRNA Library Preparation Kit (Illumina). The mRNA was purified from total RNA using oligo (dT)-conjugated magnetic beads. Purified mRNAs were fragmented at 94°C for 8 min and first strand cDNA was synthesized using random hexamer primers and SuperScriptII Reverse Transcriptase (Invitrogen) in First Strand Synthesis Act D Mix (Illumina). Second strand cDNA synthesis was subsequently performed in Second Strand Marking Master Mix (Illumina). After adenylation of the 3' ends, RNA Adapter Index codes were ligated to their respective cDNA samples. In order to select ~280bp cDNA fragments, the libraries were purified with AMPure XP system (Beckman Coulter). To enrich adaptor-ligated cDNA fragments, the libraries were PCR-amplified (PCR Master Mix and PCR Primer Cocktail, Illumina) and purified with AMPure XP system. The quality of the libraries was confirmed using Agilent High Sensitivity D1000 ScreenTape in an Agilent 2200 TapeStation System (Agilent Technologies). The cDNA concentration was measured by qPCR and with Qubit® DNA Assay Kit in a Qubit® 2.0 Fluorometer (Thermo Scientific). cDNA libraries were applied to NextSeq 500/550 Mid Output Kit v2.5 (150 cycles) and sequencing on Illumina NextSeq System to obtain 5 million of 75 bp paired-end sequencing reads per sample.