title: GSM8506136: EAE_3; Mus musculus; RNA-Seq
library_construction_protocol: The disease was induced in the SJL mice (Strain #: 000686 RRID:IMSR_JAX:000686) using proteolipid protein139-151(PLP139-151) peptide (200 μg/mouse) emulsified in Complete Freund's Adjuvant (CFA, Sigma Chemicals, St. Louis, MO, USA) supplemented with 4 mg/ml heat-killedMycobacterium tuberculosisH37Ra (400 μg; Becton, Dickinson and Company, Sparks, MD, USA). One set of mice was injected with CFA without antigen to serve as a control.It was followed by intraperitoneal treatment with 300ng MaR1 (Cayman) from day 6 till day 70. On day 70, mice were euthanized, and the spinal cord samples were sent for RNAseq (CD-Genomics) for comparative gene expression profiling between the CFA, EAE,and MaR1 treated groups.For RNA-seq, the spinal cords of three mice from each group, CFA, EAE, and MaR1-treated mice, were subjected to RNA-seq via the services of CD-Genomics, NY. RNA wasisolated using the Qiagen RNeasy Lipid Tissue Kit (Qiagen, Hilden, Germany) Isolated RNA sample quality was assessed by High Sensitivity RNA Tapestation Assay (Agilent Technologies Inc., California, USA)] and quantified by Qubit 2.0 RNA HS assay (ThermoFisher, Massachusetts, USA). Paramagnetic beads coupled with oligo d(T)25 are combined with total RNA to isolate poly(A)+ transcripts based on NEBNext® Poly(A) mRNA Magnetic Isolation Module manual (New England BioLabs Inc., Massachusetts, USA). Prior to first strand synthesis, samples are randomly primed (5´ d(N6) 3´ [N=A,C,G,T]) and fragmented based on manufacturer's recommendations. The first strand is synthesized with the Protoscript II Reverse Transcriptase with a longer extension period, approximately 30 minutes at 42⁰C. All remaining steps for library construction were performed according to the NEBNext® Ultra™ II Non-Directional RNA Library Prep Kit for Illumina® (New England BioLabs Inc., Massachusetts, USA). Final libraries quantity was assessed by Qubit 2.0 (ThermoFisher, Massachusetts, USA) and quality was assessed by TapeStation HSD1000 ScreenTape (Agilent Technologies Inc., California, USA). The final library size was about 400bp with an insert size of about 250bp. Illumina® 8-nt dual-indices were used. Equimolar pooling of libraries was performed based on QC values and sequenced on an Illumina® NovaSeq 6000 (Illumina, California, USA) with a read length configuration of 150 PE for 40 M PE reads per sample (20M in each direction).