Example 1 High-throughput sequencing and screening of differentially expressed genes
 1. Sampling
 A total of 10 patients with osteoarthritis in the Department of Orthopedics of Peking Union Medical College Hospital from October 2012 to December 2015 were collected. A total of 10 cases were collected in the case group, and a total of 4 cases were collected from patients with other diseases hospitalized in the Department of Orthopedics during the same period. Obtain cartilage tissue samples of all study subjects, and store them in a -80℃ low-temperature refrigerator after numbering.
 The case group met the diagnostic criteria of knee joint OA and had knee joint replacement surgery; the control group was patients with meniscus injury and cruciate ligament injury undergoing arthroscopic surgery. According to the 1995 diagnostic criteria for osteoarthritis of the American Rheumatism Association, patients who were diagnosed with severe osteoarthritis and indicated for knee replacement.
 Among them, the clinical diagnostic criteria are based on the standards established by the American Rheumatism Association in 1995:
 (1) Knee pain most of the time within 1 month;
 (2) Sound when joints move;
 (3) Morning stiffness is not more than 30 minutes;
 (4) Age not less than 40 years old;
 (5) Bone swelling of the knee joint with bounce;
 (6) Bone swelling of the knee joint is not accompanied by bounce.
 At least ((1), (2), (3), (4) or (1), (2), (3), (5) or (1), (6) can diagnose OA.
 2. Extract total RNA from cartilage tissue
 use Reagent (invitrogen, article number 15596-018) is used to extract RNA from the sample. The experimental operation is carried out according to the product manual. The specific operation is as follows:
 Collect the sample and freeze it in liquid nitrogen. After taking it out, place the tissue in a pre-cooled mortar for grinding. After the tissue sample is powdered:
 ①Add Trizol and store at room temperature for 5 minutes;
 ②Add 0.2 mL of chloroform, shake the centrifuge tube vigorously, mix well, and leave it at room temperature for 5-10 minutes;
 ③After high-speed centrifugation at 12000 rpm for 15 minutes, suck the upper water phase (absorb 70%) into another new centrifuge tube, taking care not to suck the protein material between the two water phases. Transfer to a new tube, add an equal volume of -20°C pre-cooled isopropanol, mix thoroughly by inversion, and place on ice for 10 minutes;
 ④Carefully discard the supernatant after leaving at 12000rpm for 15 minutes at a high speed, add 75% DEPC ethanol to wash the paint precipitate (stored at 4°C) at a ratio of 1mL/mLTrizol, wash the paint precipitate, shake and mix, and centrifuge at 12000rpm for 5 minutes at 4°C;
 ⑤ Discard the ethanol liquid, place it at room temperature for 5 minutes to fully dry the precipitate, and add DEPC-treated water to dissolve the precipitate;
 ⑥Measure the purity and concentration of RNA with Nanodrop2000 UV spectrophotometer, and store it at -80℃. RNA quality criteria: the OD260/OD280 value of the RNA sample is between 1.7-2.2; the total RNA electrophoresis pattern has clear 28S and 18S bands; the electrophoresis pattern after 1 hour incubation at 70°C and the pattern before the water bath are not obvious difference.
 3. Quality analysis of RNA samples
 After RNA extraction, agarose gel electrophoresis can be used to preliminarily determine whether the extracted RNA sample is qualified or not, and whether it can be used for further transcriptome analysis. Furthermore, the extraction of RNA samples was detected by NanoDrop1000 spectrophotometer. The sample requirements for RNA-seq sequencing: OD260/OD280 is 1.8-2.2.
 4. High-throughput sequencing
 The sequencing platform is Illumina's HiSeq2500 high-throughput sequencing platform, which performs high-throughput transcriptome deep sequencing. After sequencing, we use Fast-QC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) software The overall evaluation of the quality of the sequencing data, including the quality value distribution of bases, the location distribution of quality values, GC content, PCRduplication content, kmer frequency, etc. In the differential gene expression analysis, according to the obtained FPKM value, the internationally recognized algorithm EBSeq is used for differential screening. Among them, when screening, LOG2FC> 1 or <0.05. In order to better understand the functions of differentially expressed genes, we conducted GeneOnlogy and signal pathway analysis on differentially expressed genes, and performed functional annotation and protein interaction network analysis on differentially expressed genes. In view of the results of the above data analysis, we screened in combination with literature The differentially expressed gene SERPINB3 was down-regulated in tissues from osteoarthritis samples.