title: GSM3137228: 7D1_MO; Rattus norvegicus; RNA-Seq
library_construction_protocol: After the behavioural assessment of ischemic stroke, the rats (n=4-6) were transcardially perfused with 250 ml of cold PBS to remove blood from the brain. The brain was isolated and transported in HBSS. The ipsilateral side of the brain was dissected and subjected to mechanical and enzymatic dissociation using neural tissue dissociation kit (Milteny Biotech) according to the manufacterer's instruction. Myelin was removed using Percoll density gradient centrifugation. Briefly, single suspension of cells was mixed with the Percoll solution and centrifuged for 20 min., 950 rcf at 4°C. Cell pellet was resuspended in DPBS containing 2% BSA and counted. For staining, the cells were suspended (1 x 10^6 cells per each) and incubated with the respective antibodies at 4ºC: FITC Mouse Anti-Rat CD11b, FITC Mouse IgA Kappa Isotype control, PE-Cy 5 Mouse Anti-Rat CD45, PE-Cy 5 Mouse IgG1 Kappa Isotype control to determinate the percentages of microglia and macrophages subpopulations and analyzed using CellQuest software. Immune subpopulations were determined by flow cytometry. Quadrant gates were drawn on two cell subpopulations with differences in the surface expression of CD11b and CD45 antigen: microglia (CD11b+CD45low), blood–derived macrophages (CD11b+CD45high). All antibodies used were purchased from BD biosciences. In total, 21 strand-specific RNA libraries for high-throughput sequencing were prepared (three biological replicates for each treatment) using the KAPA Stranded mRNA Sample Preparation Kit according to the manufacturer's protocol. Briefly, the poly-A containing mRNA molecules were purified from 500ng of total RNA (extracted FACS sorted cells from the brain samples of sham operated and MCAo animals) using poly-T oligo-attached magnetic beads (Kapa Biosystems, MA, USA ) The mRNA was then fragmented and the first-strand cDNA was synthesized using reverse transcriptase and random hexamers. Second cDNA synthesis was performed by removing the RNA template and synthesizing a replacement strand, incorporating dUTP in place of dTTP, to generate double-stranded (ds) cDNA. dsDNA was then subjected to the addition of “A” bases to the 3′ ends and ligation of adapters from NEB followed by uracil digestion in a loop structure of adapter by USER enzyme from NEB (Ipswich, MA, USA). Amplification of fragments with adapters ligated on both ends was performed by PCR using primers containing Truseq barcodes from NEB (Ipswich MA, USA).
attributes.tag: GEO Accession
attributes.value: GSM3137228