GSM8626406: MCF-7 cells, sh2-ME_3, day1; Homo sapiens; RNA-Seq
Total RNA was isolated using the Trizol Reagent (Invitrogen Life Technologies), after which the concentration, quality and integrity were determined using a NanoDrop spectrophotometer (Thermo Scientific). Three micrograms of RNA were used as input material for the RNA sample preparations. Sequencing libraries were generated according to the following steps. Firstly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer. First strand cDNA was synthesized using random oligonucleotides and Super Script II. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and the enzymes were removed. After adenylation of the 3′ ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. To select cDNA fragments of the preferred 400-500 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter,Beverly, CA, USA). DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15 cycle PCR reaction. Products were purified (AMPure XP system) and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent). The sequencing library was then sequenced on NovaSeq 6000 platform (Illumina) Shanghai Personal Biotechnology Cp. Ltd. Choose a fragment with a length of 300 bp because the adapter length is fixed, and if the interrupted fragment is shorter, it will lead to a high proportion of the adapter sequence, thus reducing the proportion of valid data; If the length of the interrupted fragment is long, it is not conducive to the generation of clusters during on-machine sequencing. The first strand of cDNA was synthesized with 6 base random primers and reverse transcriptase using RNA as a template, and the second strand cDNA was synthesized using the first-strand cDNA as a template.After the library construction is completed, PCR amplification is used for library fragment enrichment, and then the library is selected according to the fragment size, and the library size is 450 bp. Next, the library was qualitatively checked on the Agilent 2100 Bioanalyzer, and the total and effective concentrations of the library were measured. Libraries containing different index sequences (each sample plus a different index, and finally the off-order data for each sample according to the index) are then mixed proportionally according to the effective concentration of the library and the amount of data required for the library. The pooled libraries are uniformly diluted to 2 nM and formed into single-stranded libraries by alkaline denaturation.After RNA extraction, purification, and library construction, the samples were sequenced using next-generation sequencing (NGS) based on the Illumina sequencing platform.