GSM3333698
GSM3333698: UBR2_Log_0hrs_c_RNA; Saccharomyces cerevisiae; RNA-Seq
mRNA extraction:  Frozen cell pellets were resuspended in 200 µL of Lysis buffer (10mM Tris [pH 8.0], 0.5% SDS, 10 mM EDTA). Following the addition of 200 µL Acid Phenol [pH 4.3], samples were vortexed for 30 seconds.  Samples were incubated at 65oC for 1 hour in a Thermomixer with intermittent shaking (2000 RPM for 1 minute every 15 minutes). 400 µL of ethanol was then added and the RNA was purified using the Direct-zolTMRNA Miniprep Plus kit (Zymo research) according to the manufacturer's protocol including the optional DNase digestion step. RNA integrity was confirmed using an Agilent Bioanalyzer. RNA-Seq Library Preparation: RNA-seq libraries were prepared first by removing rRNA with the Ribo-Zero Gold rRNA Removal Kit (Yeast) (Illumina Cat #MRZY1324 and eluted in 30 µL. After rRNA removal, RNA was bound to AGENCOURT RNACLEAN XP beads (Beckman coulter cat#A66514) using a 2X bead volume (60 µL) followed by washing and elution as per vendor's protocol (except that RNA was eluted in Fragment, Prime, Finish Mix from the TruSeq Stranded mRNA Library Prep [Illumina cat# 20020595]). Subsequent steps were carried out as per the protocol for the TruSeq Stranded mRNA Library Prep. Libraries were indexed using either Illumina barcode kit cat#s 20020492, 20020493 or 20019792). Libraries were sequenced on a hISEQ4000 (with 150x150 paired-end reads). ATAC-Seq Sample Preparation: 5 million cells were spun down (1200 rcf / 4 min. / room temperature) and resuspended in 250 mL media.  Propidium Monoazide (PMA, Qiagen 296015) was added to a concentration of 50 mM, the cells were incubated in the dark at room temperature for 10 min. and then illuminated in the BLU-V system (Qiagen 9002300) for 20 minutes at 4oC.  Cells were spun down, washed twice with SB buffer (1M Sorbitol, 40 mM HEPES [pH7.5], 10 mM MgCl2) and then resuspended in 190 mL SB Buffer. 10 µL of 10 mg/mL Zymolyase-100T was added and the cells were incubated at 30oC for 30 min. with light shaking (600 RPM) in a thermomixer (Eppendorf). Spheroplasts were spun down (1500 rcf / 5 min. / room temperature) and washed twice with SB Buffer, resuspended in 50 mL Tagmentation Mix (25 mL Nextera Tagment DNA Buffer, 22.5 mL H2O, 2.5 mL  Nextera Tagment DNA Enzyme I) and incubated at 37oC for 30min.  DNA was purified over DNA Clean & ConcentratorTM-5 kit (Zymo Research) following the manufacturer's protocol, eluted in 11ml H2O and stored at -20oC until ready for PCR.          PCR reactions were set up using Nextera Index i5 and i7 series PCR primers (Illumina).  Mixed 25ml NEBNext Hi-Fidelity 2x PCR Master Mix, 7.5ml H2O, 6.25ml i5 primer (10 mM), 6.25ml i7 primer (10 mM) and 5 mL tagmented DNA from above such that each sample has a unique barcoded primer pair. Ran PCR amplification (1 cycle: 72ºC for 5 min.; 1 cycle: 98ºC for 30 sec.; 8 cycles: 98ºC for 10 sec., 63ºC for 30 sec., 72ºC for 1 min.; hold at 4ºC).  PCR reactions were cleaned up using the Agencourt AMPure XP system (Beckman Coulter) 1st by negatively selecting against large DNA fragments using 0.4x volume of beads and then by positively selecting for the desired fragments using 1.5x volume of beads.  The final solution was re-purified over DNA Clean & ConcentratorTM-5 columns to eliminate primers, eluted in 22 µL H2O and analyzed on the BioAnalyzer.
GEO
GSM3333698
GEO Accession
GSM3333698