Genomics data for CCl4-induced mice liver fibrosis
Objective: To understand the transcriptome data of liver fibrosis induced by CCl4 in mice.Methods: C57BL/6 mice were used to duplicate the liver fibrosis model induced by CCl-4.After successful replication, mouse RNA was extracted for subsequent experiments.The basic principle of RNA extraction is to prevent RNA degradation during extraction, maximize RNA extraction efficiency, and ensure the extraction of high-quality RNA with good integrity and purity from target samples.According to the species (such as animal sample or plant sample) and sample status (such as animal tissue or animal cell) of the sample, select the appropriate extraction reagent and corresponding extraction method.The detection of high-quality RNA in samples is the basis for the success of the whole project. In order to ensure the accuracy of sequencing data, we performed quality inspection on samples, and the library construction can only be performed after the detection results meet the requirements for sequencing library construction.Sample test results are shown in the quality inspection report.After the library construction samples passed the test, the mRNA of eukaryotes was enriched with magnetic beads with Oligo (dT).Subsequently fragmentation buffer was added to randomly interrupt the mRNA.First-strand cDNA was synthesized using mRNA as a template with a six-base random primer (random hexamers), followed by buffer, dNTPs, and DNA polymerase I to synthesize the second-strand cDNA, which was subsequently purified using AMPure XP beads.Purified double-stranded cDNA was then end-repaired, A-tailed and ligated with sequencing adapters, followed by fragment size selection with AMPure XP beads, and finally PCR enrichment to obtain the final cDNA library.After library construction, insert size and effective concentration of the library were tested to ensure library quality.After passing the library test, different libraries were pooling according to the amount of data under the target and sequenced on the machine.RESULTS: RNA-seq data of CCL4-induced liver fibrosis model in mice were finally obtained in our experiment, which provided a molecular biological basis for our future study of liver fibrosis in mice. Overall design: To invesitgate the effect of CCl4 on mice liver, C57BL/6 mice were used in this study. We then perform RNA-seq of 2 conditions. Sequencing contains adapter-bearing, low-quality reads. In order to ensure the quality of subsequent analysis, it is necessary to remove adaptor sequences, filter out reads with low quality (Low Quality, the number of bases with base quality value less than or equal to 25 accounts for more than 60% of the entire reads) and N (N indicates that the base information cannot be determined) ratio greater than 5%, obtain reads.HISAT2, which can be used for subsequent analysis, is a software for second-generation sequencing sequence alignment, which can align the whole genome, transcriptome and exome data with the reference genome. Using the improved BWT algorithm, it can efficiently align the sequencing reads to the reference genome. In this project, Clean Reads were aligned to the designated genome using HISAT2 software to obtain information on their location on the reference genome. The algorithm of HISAT2 is mainly divided into three parts: (1) aligning the whole sequencing sequence to a single exon of the genome; (2) aligning the sequencing sequence segment to two exons of the genome; and (3) aligning the sequencing sequence segment to more than three (including three) exons of the genome.