GSM8519147: Line WT, Heat Shock by 5.5 hours, biol rep 1; Arabidopsis thaliana; RNA-Seq
Total RNA Sample QC. All samples need to pass through the following four steps before library construction: (1) Nanodrop: tests RNA purity (OD260/OD280). (2) Agarose Gel Electrophoresis: tests RNA degradation and potential contamination. (3) Agilent 2100: checks RNA integrity. Library Construction. After the QC procedures, mRNA was enriched using oligo(dT) beads. The mRNA was then fragmented randomly in fragmentation buffer, followed by cDNA synthesis using random hexamers and reverse transcriptase. After first-strand synthesis, a custom second-strand synthesis buffer (Illumina) is added with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand by nick-translation. The final cDNA library is ready after a round of purification, terminal repair, A-tailing, ligation of sequencing adapters, size selection and PCR enrichment. Library QC and sequencing. Library concentration was first quantified using a Qubit 2.0 fluorometer (Life Technologies), and then diluted to 1 ng/µl before checking insert size on an Agilent 2100 and quantifying to greater accuracy by quantitative PCR (Q-PCR) (library activity >2 nM). Libraries were fed into Illumina NovaSeq 6000 machines for sequencing.
GEO
GSM8519147_r1