GSM5170735: 2h_R-_2 [17446_6]; Plasmodium berghei; RNA-Seq
Complete RNA was isolated from the samples using Trizol/chloroform extraction followed by isopropanol precipitation and its concentration and integrity was verified using Agilent Bioanalyzer (RNA 6000 Nano kit) and NanoDrop 1000 spectrophotometer. 1-2μg of total RNA from each sample (all complete sample if the yield was lower) was used for mRNA isolation (Magnetic mRNA Isolation Kit, NEB). First strand cDNA synthesis was performed using the SuperScript III First-Strand Synthesis System and a 1:1 mix of Oligo(dT) and random primers (Invitrogen). The DNA/RNA hybrids were purified using Agencourt RNACleanXP beads (Beckman Coulter) and the second cDNA strand was synthesized using a 10 mM dUTP nucleotide mix, DNA Polymerase I (Invitrogen) and RNAseH (NEB) for 2.5 h at 16°C. The long cDNA fragments were purified and fragmented using a Covaris S220 system (duty cycles= 20, intensity = 5,cycles/burst =200,time= 30s). The ~200 bp long fragments were end-repaired, dA-tailed and ligated to “PCR-free” adapters (Kozarewa et al., 2009) with index tags using NEBNext kits according to the manufacturer's instructions. Excess adapters were removed by two rounds of clean-up with 1xvolume of Agencourt AMPure XP beads. Final libraries were eluted in 30μl water, quality-controlled using Agilent Bioanalyzer (High Sensitivity DNA chip) digested with USER enzyme (NEB) and quantified by qPCR. For some libraries, additional 5 cycles of PCR amplification were performed, using KAPA HiFi HotStart PCR mix and tag-specific primers to obtain enough material for sequencing. Pools of indexed libraries were sequenced using an Illumina HiSeq2500 system (100 bp paired-end reads) according to manufacturer's manual.
GEO
GSM5170735