title: GSM2092850: Ctr1_Dk_4h; Prochlorococcus marinus subsp. pastoris str. CCMP1986; Prochlorococcus phage P-HM2; RNA-Seq
library_construction_protocol: Total RNA was extracted using the Ambion mirVana RNA isolation kit, and residual genomic DNA was removed using the Turbo DNA-free kit (Ambion). RNA was then concentrated with the RNA Clean & Concentrator-5 kit (Zymo Research). Total RNA (150 ng) was fragmented to the range of 60–200 nt by magnesium-catalyzed hydrolysis (40 mM Tris-Acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate) for 4 min at 83 ∘C, and purified with the RNA Clean & Concentrator-5 kit. Strand-specific RNA-Seq libraries were prepared using a dUTP second-strand marking protocol as described previously (Levin et al., 2010). Briefly, first-strand cDNA was synthesized from fragmented RNA with random primers (Invitrogen) and Superscript III reverse transcriptase (Invitrogen). Second-strand cDNA was synthesized using dUTP instead of dTTP. Paired-end libraries for Illumina sequencing were prepared from purified double-stranded cDNA (Agencourt AMPure XP beads) as recommended by Illumina. Second strands of cDNA containing dUTP were removed with the Uracil Cleavage System (Enzymatics), and libraries were amplified with the sequencing primers. A duplex-specific nuclease (DSN)-based method (Yi et al., 2011) was used to remove the 16S and 23S rRNAs, which has been successfully used in Prochlorococcus (Giannoukos et al., 2012). Illumina sequencing primers with barcodes were used to amplify the DSN treated libraries. Barcoded libraries (40) were pooled with equal proportion in one lane and paired-end sequencing was done on an Illumina HiSeq 2000 (40 nt for insert + 6 nt for barcode).