title: GSM5194283: Raw.Sm_Bif_NFlow_rb4_BTm; Oryctolagus cuniculus; RNA-Seq
library_construction_protocol: Immediately following euthanasia, animals were perfused with 1 U/mL heparin in 0.9% saline solution for 10 minutes. Then, the brain was removed, and the most posterior cerebral arteries, from the vertebral arteries to the posterior cerebral arteries and superior cerebellar arteries, were surgically excised. The BA with the BT and short portions of the downstream branches was placed in optimal cutting temperature compound and flash frozen in liquid nitrogen. Longitudinal sections of the BA and coronal sections through the BT were cut at 8 µm thickness, adhered to nuclease-free Foil Membrane Slides for use with the Leica laser microdissection system and stored at -80oC until laser microdissection. Immediately prior to microdissection, sections were fixed for 2 min with ice-cold 70% ethanol, washed twice for 60 sec with ice-cold RNase-free water to remove residual embedding media, and dehydrated by one 2-minute wash in 95% ethanol and two 2-minute washes in 100% ethanol, then air dried for one hour in a vacuum desiccator at room temperature. Dehydrated sections were micro-dissected on a Leica LMD6000 system (Leica Microsystems): ~10 um wide strips were cut from the intimal layer (containing ECs) and separate strips were cut through the full thickness of the remaining medial layer (containing VSMCs). Intimal and medial tissue fragments from the BA and from a region extending ~100 µm distally from the apex of the BT were collected separately into dry microfuge tubes and held at -80 degrees C until RNA extraction. To increase RNA yield, tissue from ~10 sections were combined for each sample. Total RNA was extracted and amplified from each sample tube using SMART-Seq v4 Ultra Low Input RNA Kits (Takara Bio). To check the quality of extraction and amplification, an aliquot of the end product was examined by electrophoresis using an Agilent 2100 BioAnalyzer RNA 6000 Pico Chip (Agilent, Las Vegas, NV). RNA samples to be sequenced had concentrations of 3.5-40 ng/µl and a broad molecular-weight distribution spanning 100-6000 bp and peaking at 800-2000 bp. cDNA libraries were prepared using Nextera DNA Library Preparation Kits (Illumina, San Diego, CA) and subjected to 75-cycle, single-read sequencing on an Illumina NextSeq500.