Instrument: Illumina NovaSeq 6000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: At each time point, approximately 4x106 cells were pelleted with Tween-20 (0.005%, v/v) by centrifugation at 1,100 x g and 4oC for 2 min. The cell pellet was flash frozen in liquid nitrogen and stored at -80oC before processing. Total RNA was extracted with the TRIzol reagent (Thermo Fisher Scientific, Cat No. 15596026) as described before with some modifications (Zones et al., 2015). RNA was purified by RNeasy mini-column (Qiagen, Cat No. 74106) after on column digestion with RNase-free DNase (Qiagen, Cat No. 79256) according to the manufacturer's instructions. RNA was quantified with Qubit™ RNA BR Assay Kit, (Life technology, Cat No. Q10210). Total 0.4 μg RNA was reverse transcribed with oligo dT primers using SuperScript® III First-Strand Synthesis System (Life technology, Cat No. 18080-051) according to the manufacturer's instructions. Quantitative real-time PCR (RT-qPCR) analysis was carried out using a CFX384 Real-Time System (C 1000 Touch Thermal Cycler, Bio-Rad, Hercules, California) using SensiFAST SYBR No-ROS kit (Bioline, BIO-98020). PCR was set up as follows: 2 min at 95˚C, followed by 40 cycles of 5 s at 95oC, 10 s at 60oC and 15 s at 72oC. Melting curve was checked right after all PCR cycles to make sure there are no primer dimers or unspecific PCR products. Expression of G-protein β-subunit-like polypeptide CBLP (Cre06.g278222), (Schloss, 1990) remain stable among all time points, and were used as internal controls (Xie et al., 2013). The relative expressions were calculated relative to its expression in pre-heat by the ΔΔCT method (Livak and Schmittgen, 2001b). The qPCR primers for tested genes (CBLP, HSP22A, HSP90A, and others) are listed in Supplementary Table 1. PCR efficiencies were checked and were employed to the 2−ΔΔCT method to calculate the relative expression as described previously (Livak and Schmittgen, 2001; Hellemans et al., 2007; Remans et al., 2014). Three biological replicates for each time point and treatment were conducted. Library construction was perfored at the Department of Energy (DOE) Joint Genome Institute (JGI)