GSM8067751: IVD, WT_INJURED4; Mus musculus; RNA-Seq
From each mouse tail,  Co3/4, 4/5,5/6 and Co6/7 IVDs were shaved off the cartilaginous endplate with a scalpel under a dissecting microscope (VistaVision, VWR International, Radnor, PA). The IVD tissues were from all 4 coccygeal IVDs of each animal were isolate, and immersed in RNALater (Ambion, Foster City, CA) overnight and preserved at -80°C until extraction. Total cellular RNA was isolated by the Trizol method. Specifically, RNALater was eliminated, and the tissues were rapidly frozen with liquid Nitrogen before being transferred into Trizol (Invitrogen, Carlsbad, CA). Subsequently, the tissues were homogenized using a homogenizer equipped with disposable OmniTip probes designed for hard tissue (Omni International, Kennesaw, GA). RNA was then precipitated with 70% ethanol and further purified using a RNeasy Micro Kit (Qiagen). RNA Library Preparation and Sequencing was performed by Azenta US (South Plainfield, NJ). Specifically, RNA samples were quantified using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) and RNA integrity was checked with 4200 TapeStation (Agilent Technologies, Palo Alto, CA, USA). rRNA depletion sequencing library was prepared by using QIAGEN FastSelect rRNA HMR Kit (Qiagen, Hilden, Germany). RNA sequencing library preparation uses NEBNext Ultra II RNA Library Preparation Kit for Illumina by following the manufacturer's recommendations (NEB, Ipswich, MA, USA). Briefly, enriched RNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end-repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies, Palo Alto, CA, USA), and quantified using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were multiplexed and clustered on one flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq instrument according to the manufacturer's instructions. The samples were sequenced using a 2x150 Pair-End (PE) configuration Raw sequence data (.bcl files) generated from Illumina HiSeq were converted into fastq files and de-multiplexed using Illumina bcl2fastq program version 2.20. One mismatch was allowed for index sequence identification.