GSM8834048: 150SI1; Homo sapiens; RNA-Seq
Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. Total RNA was tested by Annoroad Gene Technology Corporation (Beijing) using the following methods: sample purity was tested using a NanoPhotometer® spectrophotometer (Thermo Fisher); RNA integrity and concentration were tested using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Total RNA samples that met the requirements were selected for construction of sequencing libraries. After the total RNA sample is qualified, it is purified according to the characteristics of mRNA from different sources. The 3' end of eukaryotic mRNA has a polyA tail structure, and magnetic beads with Oligo (dT) are selected for enrichment and purification; prokaryotic mRNA does not have this characteristic, and a kit is selected to remove rRNA to obtain more effective information. Fragmentation Buer is added to the purified mRNA to make its fragments into short fragments. Then, the fragmented RNA is used as a template, and random primers are used for reverse transcription to achieve the synthesis of the first strand of cDNA, and the 2nd Strand Marking Buer and 2nd Strand/End Repair Enzyme Mix are added to synthesize the second strand of cDNA. After end repair, addition of base A, addition of sequencing adapters, the target fragment is recovered by magnetic bead screening, and PCR amplification is performed to complete the entire library preparation. After the library construction was completed, Qubit3.0 was used for preliminary quantification, and the library was diluted to 1 ng/ul. Then, the insert size of the library was detected using Agilent2100. When the insert size met expectations, the effective concentration of the library was accurately quantified (library effective concentration>10nM) using Bio-RAD CFX 96 fluorescence quantitative PCR instrument and Bio-RAD KIT iQ SYBR GRN to ensure the quality of the library. The qualified library was sequenced using the Illumina platform. The sequencing strategy was PE150.
GEO
GSM8834048_r1