Role of sulfide under non photorespiratory conditions in Arabidopsis plants
Leaves from 30 days old plants were harvested and grounded under liquid nitrogen, pestle and mortar.  Arabidopsis thaliana Col-0 wild-type plants were grown at constant humidity of 70% with 16 h of light (120 µEm-2 s-1) at 20 °C and 8 h of dark at 18 °C. Plants were grown for 30 days under nonphotorespiratory conditions by automatically injecting CO2 to a final concentration of 0.7% (v/v), and harvested at 30 days (high CO2 treatment-nonphotorespiratory conditions, NPC). and other batches of plants were transferred for 3 days from high CO2 (0.7% CO2 v/v) to normal CO2 conditions (0.04% CO2 v/v) and then harvested for further analysis (active-photorespiratory conditions, APC).  For the sulfide treatment, plants were irrigated and sprayed with 200 µM NaHS every 3 days at least 5 times before harvesting the NPC samples and transferring to APC conditions. Control plants were treated with water. The total RNA was extracted from Arabidopsis plants grown under different conditions described above, NPC and APC with or without NaHS treatment, using an RNA prep Pure Plant Kit (Qiagen according to the manufacturer's instructions).  Before library construction, several analyses were performed to check RNA quantification, purification using poly-T-oligo-attached magnetic beads and integrity and purity using an Agilent 5400 analyzer. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis. Subsequent end repair, A-tailing, adapter ligation, size selection, amplification and purification were performed. Purified mRNA was used to construct sequencing library, which was checked using Qubit and real-time PCR for quantification and a bioanalyzer for size distribution detection. Clustering of the index-coded samples was performed, the library preparations were sequenced on an Illumina platform, and paired-end reads were generated.