GSM3723054: H2-HS: wild type HSF1; Saccharomyces cerevisiae; RNA-Seq
Hot phenol total RNA isolation Novogene Corp library service: After the QC procedures, mRNA from eukaryotic organisms is enriched using oligo(dT) beads. The mRNA is 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 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).