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| Status |
Public on Jan 01, 2022 |
| Title |
recovery_8h_35C_A |
| Sample type |
SRA |
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| Source name |
CC1690
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| Organism |
Chlamydomonas reinhardtii |
| Characteristics |
strain: CC1690
treatment: recovery following 35C high temperature
time point: 8h
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| Treatment protocol |
PBR temperatures were shifted to moderate or acute heat stress conditions (35oC and 40oC respectively in different PBRs) for 24 hours, then shifted back to 25oC for 48 hours for recovery.
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| Growth protocol |
Chlamydomonas reinhardtii wildtype strain (cc1690, also called 21gr, mt+) was used in all experiments. CC1690 were grown in standard Tris-acetate-phosphate (TAP) medium in 400mL photobioreactors (PBRs) (Photon System Instruments, FMT 150/400-RB). Cultures were illuminated with constant 100 µmol photons m-2 s-1 light (50% red: 50% blue), mixed by bubbling with filtered air at a flow rate of 1 L/minute. Algal growth was turbidostatically maintained using OD680 during the experiments, so the algal cultures were not limited by nutrients or light. After PBR inoculation, cultures were allowed to grow to a target cell density of 2.00x106 cells/mL corresponding to around 4.0 ug/mL chlorophyll content in log-phase growth at 25oC. The steady condition was maintained turbidostatically using OD680 for 4 days to allow cultures to adapt to steady growth conditions at 25oC before heat treatments
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| Extracted molecule |
polyA RNA |
| Extraction 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)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Samples were quality control filtered using the JGI BBDuk and BBMap pipelines
Samples were quality assessed using FastQC
Reads were mapped to the Chlamydomonas reinhardtii v5.6 genome (Merchant et al., 2007) using STAR (Dobin and Gingeras, 2015; Dobin et al., 2013) with the maximum number of mapped loci set to 1 and the maximum mismatches per read set to 1, resulting in >92% of all reads being uniquely mapped to the Chlamydomonas reinhardtii v5.6 genome.
Reads per feature were counted via HT-seq count (Anders et al., 2014).
The dataset was filtered for genes that met minimum read count cutoffs of at least 10 mapped reads in at least 10% of the samples, resulting in 15,541 genes for downstream analyses
Differential expression modeling was performed on transcripts per million (TPM) normalized read counts using a generalized linear mixed-effect model and a negative binomial distribution. Treatment time points were compared to pre-heat controls. Significant differential expression was defined as |log2 fold-change > 1, FDR < 0.05, and |(control mean TPM) – (treatment mean TPM)| ≥ 1. FDR correction was performed using the Benjamini-Hochberg method (Benjamini and Hochberg, 1995).
Genome_build: Chlamydomonas reinhardtii v5.6 genome
Supplementary_files_format_and_content: tab-delimited text files include TPM values for each Sample
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample
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| Submission date |
Aug 16, 2021 |
| Last update date |
Jan 02, 2022 |
| Contact name |
Ru Zhang |
| Organization name |
Donald Danforth Plant Science Center
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| Street address |
95 N Warson Rd
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| City |
St. Louis |
| State/province |
Missouri |
| ZIP/Postal code |
63132 |
| Country |
USA |
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| Platform ID |
GPL29113 |
| Series (1) |
| GSE182207 |
Systems-wide Analysis Revealed Shared and Unique Responses to Moderate and Acute High Temperatures in the Green Alga Chlamydomonas reinhardtii |
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| Relations |
| BioSample |
SAMN20811607 |
| SRA |
SRX11781328 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5520681_A_13_35C_TPM.txt.gz |
145.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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