title: Sequencing of the liver transcriptome of ob/ob mice after MFsCR treatment
design: Select the corresponding testing methods for quality inspection according to the requirements of samples and products.A certain amount of RNA samples are denatured at suitable temperature to open their secondary structure, and mRNA is enriched by oligo (dT) -attached magnetic beads.The reaction system is configured. After reacting at the suitable temperature for a fixed period of time, RNAs are fragmented.Prepare the first-strand synthesis reaction system, and set up the reaction program, synthesize the first- strand cDNA, prepare the second-strand synthesis reaction system, and set up the reaction program to synthesize the second-strand cDNA.After the reaction system and program are configured and set up, double-stranded cDNA fragments are subjected to end-repair, and then a single A nucleotide is added to the 3 ends of the blunt fragments. The reaction system and program for adaptor ligation are subsequently configured and set up to ligate adaptors with the cDNAs.The PCR reaction system and program are configured and set up to amplify the product.The corresponding library quality control protocol will be selected depending upon product requirements.Single-stranded PCR products are produced via denaturation. The reaction system and program for circularization are subsequently configured and set up. Single-stranded cyclized products are produced, while uncyclized linear DNA molecules are digested.Single-stranded circle DNA molecules are replicated via rolling cycle amplification, and a DNA nanoball (DNB) which contain multiple copies of DNA is generated. Sufficient quality DNBs are then loaded into patterned nanoarrays using high-intensity DNA nanochip technique and sequenced through combinatorial Probe-Anchor Synthesis (cPAS). Data filtering: The sequencing data was filtered with SOAPnuke [1] by (1) Removing reads containing sequencing adapter; (2) Removing reads whose low-quality base ratio (base quality less than or equal to 15) is more than 20%; (3) Removing reads whose unknown base ('N' base) ratio is more than 5%, afterwards clean reads were obtained and stored in FASTQ format. The subsequent analysis and data mining were performed on Dr. Tom Multi-omics Data mining system (https://biosys.bgi.com). RNA identification:Bowtie2[2] was applied to align the clean reads to the gene set, in which known and novel, coding and noncoding transcripts were included. Gene Quantification Differential Expression Analysis: Expression level of gene was calculated by RSEM (v1.3.1) [3]. The heatmap was drawn by pheatmap (v1.0.8) [4] according to the gene expression difference in different samples. Essentially, differential expression analysis was performed using the DESeq2(v1.4.5) [5] (or DEGseq[6] or PoissonDis[7])with Q value  0.05 (or FDR  0.001). Gene Annotation: To take insight to the change of phenotype, GO (http://www.geneontology.org/) and KEGG (https://www.kegg.jp/) enrichment analysis of annotated different expression gene was performed by Phyper based on Hypergeometric test. The significant levels of terms and pathways were corrected by Q value with a rigorous threshold (Q value  0.05).