title: GSM1263347: RNA-seq_t9; Saccharomyces cerevisiae; RNA-Seq
library_construction_protocol: 1 mL BY5765 cells from the cycle (about 15 OD/mL) were collected and flash frozen. The pellet was resuspended in 200 µl RNA stat-60 (Tel-Test, Friendswood, TX) and disrupted in a Mini-Beadbeater-16 (Biospec, Bartlesville, OK) broken up three times with 100 µl glass beads in 1.5 ml conical screw cap tube (USA SCIENTIFIC, 1415-8700, Ocala, FL). Supernatant was collected into a new microcentrifuge tube and 800 µl RNA stat-60 was added to repeat bead beating one more time. The supernatants were combined and sit at room temperature for 10 min. 200 µl chloroform was then added and the tube was vortexed for 15 sec and left at room temperature for 10 min. The tubes were centrifuged at 4°C, 16000 rcf for 15 min and the supernatant was transferred to a new tube. 500 µl isopropanol was added and mixed by inverting the tube several times. The mixture was incubated at room temperature for 10 min and centrifuged at 4°C, 16000 rcf for 15 min. The pellet was washed with 70% ethanol twice, air dried and resuspended in 100 µl H2O. Samples were run on the Agilent 2100 Bioanalyzer to ensure that high quality RNA was used. 4 µg total RNA was then prepared with the TruSeq™ RNA Sample Prep Kit (Illumina, San Diego, CA). mRNA was purified and fragmented before cDNA synthesis. cDNA was then end repaired and A-tailed. After adapter ligation, samples were PCR amplified and purified with AmpureXP beads (Agencourt, A63880, Brea, CA), then validated again on the Agilent 2100 Bioanalyzer.
attributes.tag: GEO Accession
attributes.value: GSM1263347