GSM7181417: C3,CLP88; Mus musculus; ssRNA-seq
RNA is extracted from cardiac tissues, followed by strict quality control of RNA samples. Quality control standards mainly include the following three aspects:(1) Agarose gel electrophoresis: analysis of RNA integrity and DNA contamination;(2) Nanodrop: initial quantification, detection of RNA concentration and purity (OD260/280 and OD260/230);(3) Agilent 2100 bioanalyzer: accurately detect RNA integrity. A strand-specific library was constructed by removing ribosomal RNA. First, ribosomal RNA was removed from total RNA, followed by the use of the RNase R enzyme. The RNA was broken into short fragments of 250 to 300 bp. The first strand of cDNA was synthesized using the fragmented RNA as a template and random oligonucleotides as primers. The RNA strands were subsequently degraded by RNase H. And in the DNA polymerase I system, the second strand of cDNA was synthesized from dNTPs (dUTP, dATP, dGTP, and dCTP). The purified double-stranded cDNA was end-repaired, A-tailed and ligated to A sequencing adaptor.And after screening with AMPure XP beads, about 350 to 400 bp cDNA was selected. The second strand of U-containing cDNA was degraded by USER enzymes, followed by final PCR amplification and library acquisition.