title: Transcriptomic analysis of peanuts under ambient and elevated CO2 across developmental stages
design: Transcriptomic analysis of peanuts under ambient and elevated CO2 across developmental stages
library_construction_protocol: Although genomic analysis of peanuts has been previously conducted using a reference transcriptome, the completion of the peanut genome and the availability of the peanut reference genome (Bertioli et al., 2016) provide new opportunities to associate physiological responses to changes in transcription. In this study, we used a peanut reference transcriptome obtained from our sample profiling from leaves. We collected leaf samples at different growth stages (R2-R8) from flowering to physiological maturity/harvest through the growing season to develop the transcriptomic profile associated with the elevated [CO2] response. All samples were placed in liquid nitrogen immediately after collection and stored at -80ºC for further analysis. The study was conducted under semiarid conditions (water stress was not a studied treatment) in the research fields of the cropping system research laboratory at USDA-ARS Lubbock, Texas.  Arachis hypogaea cv. C76-16; runner market type was planted at 2.5 cm depth at a planting density of 6 plants/m2 (60 x 103 plants ha-1) in 15 m row plots with 1.0 m row spacing. The experiment included two long-term [CO2] treatments (ambient; 400 ppm) and elevated; 650 ppm) under Canopy Evapotranspiration and Assimilation (CETA) chambers (Baker et al., 2014; Laza et al., 2021). Each treatment consisted of three replications. The chambers were placed on the plants in the field 10-15 days after sowing to ensure good stand establishment of selected areas. Peanut plants from both CO2 treatments were subjected to three water deficit stress and recovery cycles, and each water stress episode was evaluated by comparing three stages: well-watered stage before any stress, pre-water deficit (pwd), water deficit (wd), and well-watered recovery (ww rec). Details about experimental settings and chamber operations are described elsewhere (Laza et al., 2021; Laza et al., 2023).  Forty-two samples (42 leaves) were used for RNA extraction. For the acclimation response study, we used leaf samples collected at the three stages of the drought episode, including pre-water deficit stress (pwd/pws), water deficit stress (wd/ws), and well-watered/recovery (ww/rec), 3 days following irrigation. Frozen plant material (leaf) was ground using pre-cooled mortar and pestle with liquid nitrogen. Around 0.3 g (leaf) of the resulting fine powder was used to extract the total RNA using the RNeasy Sigma kit following the manufacturer's protocol. Final elution was done with preheated (64ºC) RNAse-free water. RNA quality and quantity were first monitored using the NanoDrop 2200 Spectrophotometer (Thermo-Scientific, Waltham, MA). Only samples with A260/230 > 1.9 and a concentration higher than 100 ng/µl were selected for further analysis. We checked the integrity of these samples using the tape station, looking for 28S and 18S bands.  We conducted serial dilutions of the total RNA samples (using RNAse-free water as a solvent) based on the concentrations obtained from the Qubit™ 2.0 fluorometer (Invitrogen, Life Technologies, Grand Island, NY). We diluted to 100, 25, 15, and 7.5 ng/µl for cDNA library construction and quantification using the NeoPrep following the manufacturer protocol. The 42 cDNA constructed libraries were validated by checking the insert size using the Agilent tape station 2200. The final concentration was confirmed using the Qubit™ 2.0 fluorometer.