GSM4454754: kali170; Plasmodium falciparum; RNA-Seq
Total RNA was prepared directly from the frozen pellets of parasitized erythrocytes, where approximately 1 ml of cell pellet was lysed in 7.5 ml Trizol (GIBCO) and RNA was extracted according to the manufacturer's instructions. The RNA input going into NGS sample preparation ranged from 100 pg up to 100 ng. The samples were prepared for transcriptome analysis following a previously described protocol (Broadbent, et.al., 2015) with minor modifications. Initially, samples were treated with TURBO DNase as described. Following DNase treatment, the Agencourt RNAclean SPRI beads (Beckman Coulter) were resuspended in 19 μL to modify the protocol for a low input ribosomal RNA depletion step. Ribosomal RNA was depleted following the Clontech Modified Protocol for Removal of rRNA from Small Amounts of Total RNA (100 ng) using Human/Mouse/Rat Ribo-Zero Magnetic Kit (Epicentre). The purification beads were resuspended in 21 μL to proceed with the fragmentation step as described12. The first and second strand cDNA synthesis steps were followed without modification except for the final bead purification was eluted in 55 μL. Aliquots from the DNase, Ribo-zero treatment, and fragmentation were analysed on Agilent BioAnalyzer Pico RNA chips, along with the final cDNA constructs. The purified cDNA was below detectable levels of the BioAnalyzer Pico chips. 50 μL of purified cDNA were prepared for NGS sequencing using the KAPA Hyper Prep Kit (KAPA Biosystems). The adaptor stock concentration was 300 nM for the ligation with 4 h incubation at 20°C. The USER digestion step was omitted as the Hi-Fi polymerase does not amplify uracil containing products. The number of amplification cycles for PCR was determined to be 14 based on quantification of the amount of post-ligation product with a KAPA Quant Kit for Illumina Sequencing (KAPA Biosystems). The purified amplified libraries were visualized on Agilent DNA 1000 chips. Libraries were quantified using the KAPA Quant Kit for Illumina Sequencing (KAPA Biosystems) and normalized to 2 nM.
GEO Accession
GSM4454754