title: Effect of dodecanoic acid (C12) on the expression of Synechocystis Salina LEGE 06099 genes by RNAseq transcriptomics analysis, 30 min and 6 h post supplementation.
design: Effect of dodecanoic acid (C12) on the expression of Synechocystis Salina LEGE 06099 genes by RNAseq transcriptomics analysis, 30 min and 6 h post supplementation.
library_construction_protocol: Mid-exponential cultures (OD ~0.5) were supplemented with 0.5 mM C12 dodecanoic acid (or DMSO as a control) and harvested after 30 min and 6 hours. 100 mL of culture from each flask was centrifuged at 3000 ×g for 10 minutes at 4 °C. Pellets were resuspended in 1mL RNAlater Stabilization buffer (Thermo Fisher Scientific) and incubated at room temperature for 5 min followed by 3,000g 5 min centrifugation. Pellets were stored at −80 °C until further analysis. Pellets were flash-frozen in liquid nitrogen and disrupted using a mortar and pestle. RNA extraction was carried out using the PureLink TM RNA Mini Kit  (Thermo Fisher Scientific) accordingto the manufacturer&#39;s protocol. DNA was removed using RapidOut DNA Removal Kit (Thermo Fisher Scientific); RNA-Seq library preparation was performed by Novogene. Firstly, ribosomal RNA was removed from total RNA, followed by ethanol precipitation. After fragmentation, the first strand cDNA was synthesized using random hexamer primers. During the second strand cDNA synthesis, dUTPs were replaced with dTTPs in the reaction buffer. The directional library was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. 