GSM2155365: K18Q_double_1; Saccharomyces cerevisiae; RNA-Seq
RNA Extraction was done using TRIzol (Thermo Fisher Scientific), followed by ethanol precipitation.  RNA samples were Dnase I treated using the Rneasy Kit (Qiagen). Strand-specific RNA-seq was performed as described in Levin JZ et al. (2010). Briefly, polyA RNA was isolated using Oligo(dT)25 magnetic beads (NEB). We used 200 ng of poly(A)+ RNA for each library. Zinc-ion fragmentation was done using RNA fragmentation reagent (Ambion) for 2 minutes at 70 degrees. We synthesized first-strand cDNA by adding 4 μl 5× first-strand buffer, 2 μl 100 mM DTT, 1 μl 10 mM dNTPs, 4 μg of actinomycin D, 200 U SuperScript III and 1 μl RNase-OUT (all reagents from Invitrogen), incubating at room temperature for 10 min followed by 1 h at 55 °C. We cleaned up first-strand cDNA using RNAClean XP beads (Agencourt) to remove dNTPs and resuspended in 104 μl H2O. We synthesized second-strand cDNA by adding 4 μl of 5× first-strand buffer, 2 μl of 100 mM DTT, 4 μl of 10 mM dNTPs with dTTP replaced by dUTP (Sigma), 30 μl of 5× second-strand buffer, 40 U of Escherichia coli DNA polymerase, 10 U of E. coli DNA ligase and 2 U of E. coli RNase H, and incubating at 16 °C for 2 h (remaining reagents from Invitrogen). End repair, A-tailing, and adapter ligation were done using Epicentre reagents, using 50ng of ligated adapters. We then incubated 1 U USER (NEB) with adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. We performed PCR with Phusion High-Fidelity DNA polymerase. We removed PCR primers using 1.8 volumes of AMPure beads.
GEO Accession
GSM2155365