title: GSM8879082: N2; Homo sapiens; ncRNA-Seq
library_construction_protocol: Total amounts and integrity of RNA were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA).A total amount of 2 μg RNA per sample was used as input material for the RNA sample preparations. Firstly, ribosomal RNA was removed and rRNA free residue was cleaned up by ethanol precipitation. Subsequently, the linear RNA was digested with 3U of RNase R (Epicentre, USA) per µg of RNA. The sequencing libraries were generated by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations. Briefly, fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. In the reaction buffer, dNTPs with dTTP were replaced by dUTP. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250-300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 ul USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 ° C for 15 min followed by 5 min at 95°C before PCR. Then PCR was performed with Phusion High- Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. 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 1.5 ng/ul, and the insert size of the library is detected by 5Agilent 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 2 nM) 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).
identifiers.namespace: GEO
identifiers.id: GSM8879082_r1