DNBSEQ-G400 paired end sequencing; Transcriptional response of Saccharomyces cerevisiae to the proapoptotic unsaturated fatty aldehyde t-2-hexadecenal by RNAseq
Transcriptional response of Saccharomyces cerevisiae to the proapoptotic unsaturated fatty aldehyde t-2-hexadecenal by RNAseq
After the respective treatment, cells were collected by centrifugation, washed with ice-cold water, and frozen in liquid nitrogen. hfd1∆ cells from a saturated preculture in YPD were inoculated at an OD600 0.1 in 30 mL of YPD medium 2% glucose and grown for 6 hours to OD600 0.8. t-2-hexadecenal (t-2-hex), dissolved in DMSO, was added from the stock solution (4.2 mM) to achieve a final concentration of 100 µM. An equal volume of DMSO (720 µL) was used for each of the control samples. The cells were incubated with DMSO for 1.5 hours and with t-2-hex for 3 hours. Total RNA extraction was performed using the acid phenol method. Cell pellets were resuspended in 400µL of TES buffer (10mM Tris/HCl pH 7.5, 10mM EDTA, 0.5% SDS) and an equal volume of acid phenol mix. The mixture was incubated at 65ºC for 45min. After 5 min on ice, phases were separated by centrifugation (1min, 10000rpm), and the aqueous phase was transferred to a new tube for another extraction with acid phenol. Again, the aqueous phase was transferred to another tube containing 40µL of 3M NaAcetate pH 5.0 and 2.5 volumes of 100% ethanol. Finally, RNA was pelleted by centrifugation at 14000rpm for 10min, washed once with 70% ethanol, and resuspended in 100µL of Milli-Q water. Following extraction, 50µg of total RNA were purified using RNeasy MiniKit (QIAGEN) according to the manufacturer's instructions. RNA integrity was assessed using a 1% agarose gel. Samples were submitted to BGI Genomics (Hong Kong) for quality control and subsequent sequencing. According to company pipeline, RNA integrity was evaluated using the Agilent Bioanalyzer 2100 (Agilent Technologies) and libraries constructed following DNBSEQ Eukaryotic Stranded Transcriptome library preparation protocol.  First, mRNA enrichment and purification were performed using oligo dT selection, which involved the use of oligo dT beads to selectively enrich mRNA molecules that possess a poly A tail. Following mRNA enrichment, RNA fragmentation was carried out to generate smaller fragments for downstream processing. Next, reverse transcription was performed using random N6-primed reverse transcription, resulting in the synthesis of first-strand cDNA. To generate the second strand of cDNA, a second-strand synthesis reaction was conducted using dUTP instead of dTTP. The synthesized cDNA was subjected to end-repair and then was 3' adenylated. Adaptors were ligated to the ends of these 3' adenylated cDNA fragments; Prior to PCR amplification, the dUTP-marked strand is selectively degraded by Uracil-DNA-Glycosylase(UDG). The remaining strand is amplified to generate a cDNA library suitable for sequencing. Many rounds of PCR amplification were performed to enrich the purified cDNA template using PCR primer; After PCR amplification, the resulting double-stranded DNA was denatured by heat, leading to the separation of the two strands. Subsequently, the single-stranded DNA was cyclized using a splint oligo and DNA ligase. Finally, DNA nanoballs were synthesized from the cyclized DNA.