GSM6735424: LPS; Rattus norvegicus; RNA-Seq
Animals were anesthetized with Ketamine/Xylazine and transcardially perfused with ice cold TBS before brain removal. Choroid plexi from bilateral lateral ventricles were microdissected in ice cold HBSS with 30uM Actinomycin D (ActD, Sigma, cat#A1410 ) and digested at 37oC for 20 min with Collagenases-I and -IV (10 U/mL, 400 U/mL final, Worthington, Cat# LS004194, and LS004184) and DNase I (30 U/mL, Roche, cat# 04716728001) and ActD (3uM). For Flow sorting, the cell suspension was blocked with CD16-CD32 antibodies (1:100, biolegend, cat#101302) and stained 30 min at 4oC with anti-rat CD45-PE/cy7 (1:100, biolegend, cat#202207) to and DAPI (50ug/mL). Cells were subsequently washed in FACS staining buffer (0.5% BSA, 2mM EDTA in PBS without Calcium/Magnesium) and centrifuged at 350 rcf for 5 minutes. Cells were resuspended in 300ul of FACS buffer and sorted by CD45 positivity on a BD ARIA instrument. CD45 positive and CD45 negative cells were collected in 0.4% BSA/PBS and 3uM ActD and sent for 10X 3prime library preparation and sequencing at Yale Center for Genome Analysis Sequencing libraries were loaded on an Illumina HiSeq4000 flow cell with sequencing settings following the recommendations of 10x Genomics (read 1: 26 cycles; read 2: 98 cycles; index i7: eight cycles; index i5: no cycles; 2.1 pM loading concentration). The Cell Ranger pipeline was designed to perform sample demultiplexing and to generate FASTQ files for read 1, read 2 and the i7 sample index. Read 2, containing the cDNA, was mapped to the reference genome (mouse mm10) using STAR