GSM7295156: SKM-1, BOR, SAMPLE6; Homo sapiens; RNA-Seq
Total amounts and integrity of RNA were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA) Total RNA was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA by using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase, then use RNaseH to degrade the RNA. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and dNTP. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR amplification, the PCR product was purified by AMPure XP beads, and the library was finally obtained. In order to ensure the quality of the library, the library needs to be tested. After the construction of the library, the library was initially quantified by Qubit2.0 Fluorometer, then diluted to 1.5ng/ul, and the insert size of the library is detected by Agilent 2100 bioanalyzer. After insert size meets the expectation, qRT-PCR is used to accurately quantify the effective concentration of the library (the effective concentration of the library is higher than that of 2nM) to ensure the quality of the library After the library is qualified, the different libraries are pooling according to the effective concentration and the target amount of data off the machine, then being sequenced by the Illumina NovaSeq 6000. The end reading of 150bp pairing is generated. The basic principle of sequencing is to synthesize and sequence at the same time (Sequencing by Synthesis). Four fluorescent labeled dNTP, DNA polymerase and splice primers were added to the sequenced flowcell and amplified. When the sequence cluster extends the complementary chain, each dNTP labeled by fluorescence can release the corresponding fluorescence. The sequencer captures the fluorescence signal and converts the optical signal into the sequencing peak by computer software, so as to obtain the sequence information of the fragment to be tested.