Instrument: Illumina HiSeq 2000
Strategy: ncRNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: We centrifuged 50 mL aliquots of harvested culture at 2400 rpm in a Sorval RT6000B to obtain at least 2 mL of packed RBCs per time-point. For time-course 1, we stored packed RBCs in 15 mL of Buffer RLT (with BME added) at -80°C prior to RNA extraction. For time-course 2, we lysed packed RBCs using a .05% saponin solution, washed liberated parasites using phosphate-buffered saline (pH 7.4), pelleted parasites at 13.2 RPM in a micro-centrifuge, and stored parasites in 1 mL of TRIZOL reagent at -80°C prior to RNA extraction. For time-course 1, we thawed RBC samples stored in Buffer RLT, added one volume of 70% ethanol, and immediately loaded the mixture onto RNeasy Midi columns (Qiagen). For time-course 2, we thawed parasite samples stored in TRIZOL reagent, performed TRIZOL-chloroform extraction, and immediately applied the aqueous layer to RNeasy Mini columns (Qiagen). During both RNeasy Midi and Mini RNA extraction procedures, we performed the optional on-column DNase I digestion for thirty minutes to remove genomic DNA. We stored isolated total RNA aliquots at -80°C with 1 unit/uL RNaseOUT (Invitrogen), and validated RNA quality using an Agilent Bioanalyzer RNA 6000 Pico Kit. We began library preparation with a second DNase treatment (Ambion TURBO DNase) using 20 units of SUPERase-In (Ambion) and 40 units of RNaseOUT (Invitrogen) to protect RNA from degradation. Each DNase reaction was incubated at 25°C for 30 minutes followed by 1.8X RNAclean SPRI bead purification (Agencourt). Second, we used a Human/Mouse/Rat Ribo-Zero Magnetic Kit (Epicentre) to deplete 18S and 28S rRNA from DNase-treated total RNA. We used 3.5-5 ug of DNase-treated total RNA for all samples except T6, T14, and TT8. In these cases, we used .4 ug, 1 ug, and .7 ug of DNase-treated total RNA, respectively. Furthermore, for T6 we mixed 4 and 8 hpi total RNA 1:1, and for T14 we mixed 12 and 16 hpi total RNA 1:1. Third, we fragmented rRNA-depleted RNA at 85°C for 8 minutes using Mg2+ Fragmentation Buffer (New England Biolabs), followed by 1.8X RNAclean SPRI bead purification (Agencourt). Fourth, we reverse-transcribed the fragmented RNA using SuperScript III (Invitrogen), 200 ng/uL of freshly prepared Actinomycin D (Sigma-Aldrich), 3 ug of 76% AT-biased random hexamers (Integrated DNA Technologies), and a gradually ramping up thermocycler program. Specifically, we set the ramp speed of a PTC-225 DNA Engine Tetrad (MJ Research) to .1°C/second, and used the following program: 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C for 5 minutes each, 42°C for 30 minutes, 45°C, 50°C, 55°C for 10 minutes each. We cleaned up first strand synthesis (FSS) reactions using both Micro Bio-Spin P-30 Rnase-free columns (Bio-Rad) and 1.8X RNAclean SPRI bead purification (Agencourt). Fourth, we performed second strand synthesis (SSS) using a biased dACG-TP/dU-TP mix (Fermentas), 10 units of E. coli DNA ligase (Invitrogen), 160 units of E. coli DNA polymerase (Invitrogen), and 2 units of E. coli RNase H (Invitrogen), followed by 1.8X AmpureXP SPRI bead purification (Agencourt). Fifth, we used an Illumina series KAPA Library Preparation Kit (Kapa Biosystems) and barcoded Y-adapters developed by the Broad Institute to end repair, A-tail, and ligate adapters to each library. We added adapters in approximately 15-fold excess of library targets, and removed un-ligated adapters and adapter-dimers using 1.0X AmpureXP SPRI bead purification (Agencourt). Sixth, we digested the dUTP-marked second strand at 37°C for 30 minutes, followed by 25°C for 15 minutes, using Uracil-Specific Excision Reagent (USER) enzyme (New England Biolabs). Seventh, we amplified libraries for as few cycles as necessary using a KAPA Real-Time PCR Library Amplification Kit (Kapa Biosystems) and PCR primers developed by the Broad Institute. Each library except for T6, T14, and TT8 required only four PCR cycles, while T14 and TT8 required eight PCR cycles, and T6 required twelve PCR cycles. Following a 2 minute denaturation step at 98°C, we cycled libraries using an ABI 7900 Real-Time PCR machine and the following 2-step program: (1) denaturation at 98°C for 20 seconds, (2) annealing and extension at 55°C for 190 seconds. Finally, we quantified libraries using a KAPA Library Quantification Kit (Kapa Biosystems) and combined barcoded libraries into two pools. We sequenced each pool on an Illumina Hiseq machine (one lane per pool) using 101-bp, paired-end read technology. We prepared the fifteen libraries used in this study in parallel (except for real-time amplification).