title: GSM8238206: SedSeq~tg_20~L82~Azide_0.5%_pH6.8~BY4741~30C~30min~Azide_0.5%_pH6.8~BR1~Sup; Saccharomyces cerevisiae; OTHER
library_name: GSM8238206
library_construction_protocol: Biochemical fractionation was completed similarly to Wallace et al. [2015], with the major exception that 20,000 g for 10 min was used rather than the original 100,000 g for 20 min. In short, 50 mL cultures of treated yeast were harvested by centrifugation at 3000 g for 5 minutes, then resuspended in 100 ¬µL of soluble protein buffer (SPB: 20 mM HEPES, pH 7.4, 140 mM KCl, 2 mM EDTA, 0.1 mM TCEP, 1:200 protease inhibitor (Millipore #539136), 1:1000 SUPERase‚Ä¢In RNase Inhibitor (Invitrogen #AM2696)), and flash frozen in liquid nitrogen as a pellet in a 2 mL Eppendorf Safe-Lock tube (Eppendorf #0030123620) with a 7 mm steel ball (Retsch #05.368.0035). The cells were then lysed using a Retsch MM400 for 5x90s at 30 Hz, chilling in liquid nitrogen between each shaking repeat. The lysed cells were resuspended in 600 ¬µL of SPB, and 100 ¬µL of total sample was transferred to 300 ¬µL of Trizol LS (Invitrogen #10296010). The remainder was centrifuged for 30 seconds at 3000 g, and 300 ¬µL of clarified lysate was transferred to a new 1.5 mL tube. This was then centrifuged for 10 minutes at 20,000 g. A 100 ¬µL supernatant sample was transferred to 300 ¬µL of Trizol LS, and 400 ¬µL of SPB was added to the pellet as a wash. After another spin at 20,000 g for 10 minutes, the supernatant was removed and the pellet was resuspended by vortexing for 15 minutes in 300 ¬µL of Trizol LS and 100 ¬µL of water. If required, 1 ng of spike-in nanoluc transcript was added to each sample at this step before RNA was isolated using Zymo Direct-Zol RNA extraction columns (Zymo #R2052), and RNA integrity was assessed by the appearance of two sharp rRNA bands on a 1% agarose gel and quantified using the absorbance at 260 nm. Sequencing libraries were prepared by from DNase I treated RNA using Qiagen FastSelect (Qiagen #334215), NEBNext Multiplex Oligos (UMI Adaptor RNA Set 1, NEB #E7335L) and NEBnext Ultra II Directional RNA library prep kits (NEB #E7760L).  Paired end 200 bp sequencing with additional reads for dual 8/8 indices plus the 11nt UMI after the i7 index was performed on an Illumina NovaSeq 6000 at the University of Chicago Genomics Facility. In general, cells were first lysed by cryo-grinding with a steel ball. When appropriate, fractionation by sedimentation or sucrose gradients was performed and RNA was dissolved in Trizol. The RNA was then purified using Zymo Direct-Zol columns. For more details, please see the protocol associated with each sample. In general, RNA sequencing libraries were prepared using either Illumina RNA sequencing kits or NEBNext Ultra II RNA sequencing kits.