GSM6410820: BT474 cell line treated with 1 μM of BETi (JQ1) for 24 hours sample 1; Homo sapiens; RNA-Seq
Total RNA was isolated and purified using the RNeasy Plus Mini Kit (Qiagen). After quantification with Qubit 2.0 (Life Technologies), the samples passed through quality control steps for sample integrity and purity with Agilent 2100 and Agarose Gel Electrophoresis. After the sample QC, the cDNA library is constructed using the NEBNext Ultra II RNA Library Prep Kit (NEB) as follows: mRNA is enriched using oligo(dT) beads. The mRNA is then fragmented via sonication in fragmentation buffer, followed by cDNA synthesis using random hexamers and reverse transcriptase. After first-strand synthesis, a custom second strand synthesis buffer and enzyme mix are added to generate the second strand by nick translation. The final cDNA library is ready after a round of DNA purification, end-repair, A-tailing, ligation of sequencing adapters, size selection and PCR enrichment. For library QC, library concentration was quantified using a Qubit 2.0 fluorometer (Life Technologies) and then diluted to 1 ng/μl before checking insert size on an Agilent 2100. Libraries were then quantified to greater accuracy by quantitative PCR (Q-PCR) prior to sequencing. RNA libraries were sequenced with 20M paired-end 150bp reads on the Illumina NovaSeq 6000 platform (Novogene Inc.).