title: GSM4712776: HiSeq_Method1 ring parasites enriched; Plasmodium falciparum; RNA-Seq
library_construction_protocol: Parasite total RNA was obtained using Trizol reagent according to the manufacturer's instructions (Invitrogen). Purified total RNA was stored in 75% ethanol at -80°C prior to use. On the day of library preparation, total RNA was dried and then resuspended in nuclease-free water. Total RNA concentration was estimated by Nanodrop ND1000 measurement assuming A260 = 1.0 is equivalent to 40 μg/mL RNA. Up to 100 μg of total RNA was used for mRNA enrichment using a Dynabeads oligo dT25 mRNA kit (Thermo), which enriches for mRNA with oligo dT magnetic beads. The oligo dT-enriched mRNA was then further enriched for 5′ capped mRNA by treatment with 1 unit of XRN-1 nuclease (New England Biolabs) for 30 min at 37°C. XRN-1 enriched mRNA was purified by acidic phenol:chloroform extraction and ethanol precipitation. The mRNA was redissolved in 20 μL of nuclease-free water and genomic DNA was removed using a Turbo DNA-free kit (Ambion). [5' adapter ligation method: method1] A ribo-G tail of was added to the purified single-stranded cDNA primed with RTNGS1 using terminal transferase (TdT, New England Biolabs). The tail length is limited to three or four Gs (Schmidt and Mueller, 1996). The TdT tailing reactions contained 2 mM GTP, 0.25 mM CoCl2, 1 unit of TdT enzyme in 1x TdT enzyme buffer and first-strand cDNA. TdT reactions were performed in 20 μL reaction volumes for 20 min at 37°C. Ribo-tailed cDNA was purified with an equal volume of Agencourt® AMPure® XP beads (Beckman Coulter) and recovered from beads in 20 μL of nuclease-free water. Double-stranded DNA adapter was made by combining 4 nmol each of NGS1 and NGS1COMP oligonucleotides in a volume of 100 μL with 10 mM NaCl and 10 mM Tris pH 8.0. The NGS1 oligonucleotide is 5′-phosphorylated to act as a donor in ligation and blocked with a three-carbon spacer at the 3′ end to prevent concatamerization of adapters. Annealing was accomplished by heating the mixture for 3 min at 80°C followed by slow cooling to 25°C (-0.1°C/min). Double-stranded DNA adapter was ligated to first-strand cDNA using T4 RNA ligase 2 (RNL2, New England Biolabs). The ligation reactions contained 40 pmol of DNA adapter, 7.5% (w/v) PEG6000, 1x RNL2 enzyme buffer, 2.5 units of RNL2 enzyme and first strand cDNA in a volume of 20 μL. The ligation reactions were performed for 99 cycles of 37°C for 30 s, 22°C for 30 s. After the completion of the RNL2 adapter ligation reactions, cDNA was diluted to 100 μL with nuclease-free water and purified with an equal volume of Agencourt® AMPure® XP beads (Beckman Coulter). Purified cDNA was recovered in 50 μL of nuclease-free water. [5' adapter ligation method: method2 ] Purified first strand cDNA primed with RTCIRC in which RNA had been removed by alkaline treatment was circularized with 100 U of CircLigaseII enzyme, 2.5 mM MnCl2, 1 M betaine, and 1x CircLigaseII buffer in a reaction volume of 20 μL as recommended by the manufacturer (Epicentre Biotechnologies). The reaction was incubated at 60°C for 1 h and the reaction terminated by heating to 80°C for 10 min. The reaction was diluted to 100 μL with nuclease-free water and cDNA purified using half the volume of Agencourt® AMPure XP beads (Beckman Coulter). Purified cDNA was recovered in 50 μL of nuclease-free water. Synthesis of cDNA:  In vitro synthesized spike-in RNA SIRV-Set 3 (Iso Mix E0/ERCC, Lexogen) was 5′ capped with Vaccinia capping system (New England Biolabs) following the manufacturer's instructions. A 100–200 ng sample of parasite mRNA (estimated by Nanodrop measurement) was mixed with 1.5 ng of SIRV-Set 3 RNA. 5′ capped SIRV-Set 3 RNA was added to samples of ring and trophozoite stage parasite mRNA. The mRNA/SIRV-Set 3 RNA mixture was fragmented by heating at 94°C for 3 min in 1x first-strand cDNA synthesis buffer supplied with SuperscriptIV reverse transcriptase enzyme (Invitrogen). Fragmented mRNA was chilled on ice for 5 min prior to reverse-transcription with 50 pmol of 5′ tagged random primer (RTNGS1 (Table S1) for adapter ligation method 1, RTCIRC (Table S1) for adapter ligation method2; see below) with SuperscriptIV as described by the manufacturer in a final volume of 20 μL. After addition of SuperscriptIV enzyme, reactions were heated to 25°C for 5 min, followed by heating to 37°C for 30 min, 50°C for 30 min, and then 70°C for 15 min. First-strand cDNA was diluted to 100 μL with 10 mM Tris 1 mM EDTA and digested with 1 μL of RNaseA/T1 mix (Thermo Scientific) for 10 min at 37°C to remove incompletely reverse-transcribed RNA. The mRNA/cDNA hybrid was purified using an equal volume of Agencourt® AMPure® XP beads (Beckman Coulter) and recovered from beads in 100 μL nuclease-free water. A 20 μL sample of purified mRNA/cDNA was kept for processing as the unenriched control sample. Adapter-ligated cDNA was used as a template for PCR amplification to make sequencing library. PCRs contained 2.5 pmol each of PE1 and ScriptSeq primers (Table S1), 200 µM dNTPs, 0.625 U PrimeSTAR® GXL DNA polymerase (Takara) and 1x buffer supplied with enzyme in a reaction volume of 25 μL. The PCR program used was 98°C for 30 s, followed by 18-25 cycles of 98°C for 10 s, 68°C for 60 s and a final extension of 68°C for 5 min. PCR products were separated in 1.5% agarose gel. DNA fragments 400-600 bp in size were excised from the gel and purified using a MinElute gel extraction kit (Qiagen), with the modification that agarose was solubilized in QG buffer at room temperature. DNA was quantified using a  Qubit™ dsDNA HS Assay kit (Invitrogen).  Libraries were pooled in equimolar ratios according to the ScriptSeq index primer recommendations (Illumina). Pooled libraries were submitted to Novogene AIT (Singapore) for standard sequencing on a HiSeq2000 instrument (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM4712776