GSM5076099: CTRL2; Rattus norvegicus; RNA-Seq
Total RNA was extracted using RNAeasy Mini Kit (Qiagen), according to the company's recommendations (a detailed protocol can be found on the producers website). Briefly, cells from the 6-well plates were disrupted in Buffer RLT and transferred to the RNAse-free tube. RNA was then purified on the spin column, including DNase I step, and eluted with 30 μL of nuclease-free water. Purity and concentration of RNA were measured using  NanoDrop One (Thermo Scientific). In total, 6 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).