GSM2752834: MiSeq_Method1 schizont parasites unenriched; Plasmodium falciparum; RNA-Seq
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 by ethanol precipitation. Double-stranded DNA adapter was made by combining 80 pmol each of NGS1 and NGS1comp oligonucleotides in a volume of 200 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). This enzyme preferentially ligates a 5′-phosphate donor from a double-stranded RNA or DNA to an RNA acceptor (Bullard and Bowater, 2006). Therefore, it should ligate double-stranded DNA adapter preferentially to ribo-G tailed cDNA. The ligation reactions contained 20 pmol of DNA adapter, 7.5% (w/v) PEG6000, 1x RNL2 enzyme buffer, 0.25 μL (units) of RNL2 enzyme and first strand cDNA. 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 purified using 25 μL Streptavidin M280 magnetic beads following the manufacturer's recommendations (Invitrogen). Synthesis of second-strand cDNA was performed on the beads using T7 DNA polymerase following the manufacturer's recommendations (New England Biolabs). The beads were resuspended in 50 μL of T7 reaction mix (0.3 mM dNTPs, 2.5 units T7 DNA polymerase, 1x T7 DNA polymerase buffer). The reaction was performed for 15 min at 37°C.  The reaction was terminated by washing the beads in 0.4 ml of washing solution (40 mM Tris pH 8.0, 10 mM MgCl2).The second-strand cDNA was eluted from the beads by heating for 3 min at 99°C in a suspension of 50 μL 1x Sodium Saline Citrate (150 mM sodium chloride, 15 mM trisodium citrate pH 7.0). [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 ligated cDNA purified using 0.5x volume of AMPure beads (Beckman Coulter). cDNA synthesis: A sample of approximately 1 µg of mRNA (estimated by Nanodrop measurement) was fragmented by heating at 94°C for 5 min in 1x first-strand cDNA synthesis buffer supplied with SuperscriptIII reverse transcriptase enzyme (Invitrogen). Fragmented mRNA was chilled on ice prior to reverse-transcription with 50 pmol of 5′ tagged random primer (RTNGS1 oligo for 5′ adapter ligation method 1, RTCIRC oligo for 5′ adapter ligation method2) with SuperscriptIII as described by the manufacturer in a final volume of 20 μL. Method 1 reverse transcription reactions also contained 40 μM biotin-11-dUTP (Thermo Scientific) to label first-strand cDNA with biotin. After addition of SuperscriptIII enzyme, reactions were heated to 25°C for 5 min, followed by heating to 37°C for 30 min, followed by 50°C for 30 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 25°C to remove incompletely reverse-transcribed RNA. The mRNA/cDNA hybrid was purified by proteinase K treatment, phenol:chloroform extraction and desalting on a Amicon-30 column (Ambion). The volume of desalted mRNA/cDNA hybrid was adjusted to 100 μL with nuclease-free water. A 20 μL sample of desalted mRNA/cDNA was kept for processing as the unenriched control sample. Library purification and Illumina sequencing: Adapter-ligated cDNA was used as a template for PCR amplification make sequencing library DNA. PCRs contained 12.5 pmol each of PE1 and Scriptseq primers, 200 µM dNTPs, 2 mM MgCl2, 0.5 units of Platinum taq enzyme (Invitrogen) and 1x Platinum taq buffer in a reaction volume of 50 μL. The PCR program used was 94°C for 90 s, followed by 18-25 cycles of 94°C for 10 s, 60°C for 60 s. PCR products were separated in 1.5% agarose gel, and DNA molecules 400-600 bp in size were excised from the gel and purified using a MinElute kit (Qiagen). DNA was quantified using a QuantIT HS kit (Invitrogen).  Libraries were pooled in equimolar ratios according to the ScriptSeq index primer recommendations (Illumina). Pooled libraries were processed with 1% Illumina phiX174 control and loaded onto MiSeq flow cells at 10 pM. 150 bp paired-end sequencing was performed using standard sequencing primers as recommended by the manufacturer (Illumina). For sequencing of libraries prepared from cDNA ligated to 5′ adapter using method1, the sequencing “recipe” for the MiSeq instrument was modified to perform the first four sequencing cycles as “dark”, meaning no data are recorded for the purpose of identifying sequencing clusters. This modification was necessary since the G-tail added by TdT during library construction provides insufficient diversity for generation of high density clusters. Without this modification, there will be loss of data in the cluster identification step (clusters passing filter quality control) of Illumina sequencing.
GEO Accession
GSM2752834