Instrument: Illumina HiSeq 4000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: 1) Brain region dissection: Brains were taken out of a minus 80 degree freezer and cryosectioned into 60um sections, which were mounted onto RNase-free glass slides. Slides were stored in minus 80 degree until dissection. During dissection, slides were placed on a minus 20 degree cold plate. One drop (approximately 50 ul) of RNAlater was placed on the brain region of interest. Each brain region then was dissected out under a dissecting video camera by using a pair of fine tipped forceps with the assistance of an 18 gauge needle with a bent tip. Bilateral tissue of the same brain region from each rat was immediately transferred into 350 ul Buffer RLT (containing beta-mercaptoethanol) and placed on dry ice. Tissue was stored in minus 80 degree before RNA extraction. 2) RNA extraction: Tissue was thawed on ice and homogenized by using a clean stainless steel bead using Qiagen TissueLyser (40Hz, 3 min). AllPrep DNA/RNA mini kit (Qiagen) was used to extract RNA. Samples were processed by using the QIAcube robot following standard protocols. The optional DNase digestion step was included for RNA samples. RNA libraries were prepared for sequencing using standard Illumina protocols.