GSM2155440: K36Q_H3Y_5; 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). 3' end RNA-seq was performed as follows. Briefly, 0.5 μg total RNA was used for each library.  Reverse transcription was performed using SmartScribe Reverse Transcriptase (Clontech).  First, RNA was incubated with 1 μl oligo dT primer (containing sequence for the PCR primer to prime off of, followed by a barcode then by poly dT sequence) for 3 minutes at 72 degrees.  Reverse transcription was done using 2 μl 5x buffer, 0.7 μl dNTP mix, 1 μl 20 mM DTT, 0.3 μl 100mM MgCl2, and 1 μl SmartScribe RT enzyme, incubating at 42 degrees for 1 hour, followed by 70 degrees for 15 minutes.  Samples were pooled and cleaned up using AmpureXP beads (Agencourt). After this step, the equivalent of 1 sample of cDNA resulting from 0.5 μg of total RNA was used for the following steps (i.e. if we pooled 10 samples together, we then used 1/10 of the resulting cDNA for the remaining steps). We then performed tagmentation using Nextera DNA kit (Illumina) at 55 degrees for 8 minutes using 0.5 μl Tagment DNA enzyme.  We then added 5 μl 0.2% SDS to stop the tagmentation reaction.  2x bead cleanup was done using AmpureXP beads (Agencourt).  PCR with one primer specific to the tagmentation- applied adapter and one primer specific to the poly-dT primer was done using HiFi KAPA polymerase for 12 cycles, followed by cleanup using 0.8X AMPure beads.
GEO Accession
GSM2155440