GSM6592522: Control2, biol rep3; Saccharomyces cerevisiae; RNA-Seq
The total RNA from each sample was extracted by hot phenol method. An oligo(dT) primer containing a VN-anker, a uridine, the sequence of the sequencing adapter, and biotin (/5BiosG/CAGACGTGTGCTCTTCCGATCTTTTTTTTrUTT TTTTTTVN) was attached to streptavidin-coated magnetic beads (Invitrogen, M280). Total RNA was incubated for 5 min at 65°C, followed by incubation with the coated magnetic beads for 10 min at 45°C. First strand synthesis was carried out using SuperScript III reverse transcriptase (Invitrogen) at 50°C. The enzyme was inactivated by incubation for 15 min at 70°C. Second strand synthesis was carried out using second strand synthesis buffer (Invitrogen), dNTPs, DNA polymerase I (New England Biolabs), Escherichia coli ligase (New England Biolabs), and RNase H (Invitrogen). To exchange the buffers and remove enzymes from the previous step, we treated the samples with Pronase (Roche). We introduced a nick at the position of the uridine with RNase HII (New England Biolabs). Then, nick translation was carried out with DNA polymerase I (New England Biolabs) and dNTPs for 8 min at 8°C. The reaction was stopped by the addition of EDTA. At the new position of the nick, we created a blunt end using T7 exonuclease, mung bean nuclease, and Klenow enzyme (New England Biolabs). Previously annealed Illumina TruSeq sequencing adapters (Ad1, 59-AATGATACGGCGACCACCGAGATC TACACTCTTTCCCTACACGACGCTCTTCCGATCT-39;and Ad2, 59-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT AGATCTCGGTGGTCGCCGTAT-39) were then blunt-end-ligated. The magnetic beads were suspended in 39 mLofH2O. Five microliters of beads was used in a PCR reaction. Ten to 13 cycles of PCR were carried out with Phusion polymerase (New England Biolabs), a forward primer (59-AATGATACGGCGACCACCGA GATC-3), and one of the Illumina TruSeq barcode reverse primers. For preparation of the library, six PCR reactions were performed and run on 8% TBE gels (Invitrogen), and the smear between 160 bp and 220 bp was cut out, gel-extracted, ethanol-precipitated, analyzed by Bioanalyzer (Agilent), and sequenced on Illumina HiSeq machines. 3'-seq