RNAseq of ipsilateral mouse brain homogenates 1 day following collagenase-induced ICH vs naive controls
RNAseq of ipsilateral mouse brain homogenates 1 day following collagenase-induced ICH vs naive controls
Mice were PBS perfused and right (ipsilateral) hemispheres were extracted and homogenised using lysing matrix D tubes (MPBio) in a Qiagen Tissue Lyser II. A model of intraparenchymal brain haemorrhage was used that created an active bleed within the brain by using collagenase VII-S (Sigma, C2399) to break down the basal lamina of cerebrovascular beds. To do this, mice were initially induced under anaesthesia using 4 % isoflurane in 70 % N2O and 30 % O2 and fur was shaven from the head. Mice were then transferred to a feedback-controlled heating pad set to 37 ˚C, securely fixed to a stereotactic frame (Stoetling), anaesthesia maintained at 1.5-2.0 % isoflurane in 30 % N2O and 70 % O2 using a nose cone, and Videne (EcoLab) applied to the scalp. A longitudinal midline incision above the skull was performed and periosteum stripped away from the midline on both sides. A burr-hole was created using a micro-drill and a glass micropipette (BLAUBRAND, pulled at 70 ˚C) inserted at the following co-ordinates from bregma: anterior-posterior 0.0 mm, lateral -2.0 mm, deep -2.7 mm. 0.5 µL of 0.09 units µL-1 of collagenase dissolved in saline was then injected at a rate of 1 µL min-1. The needle was left in situ for 10 min before removal and wounds sutured. The scalp was cleaned once more with Videne and a topical analgesia was applied (EMLA cream, AstraZeneca) before animals were given a subcutaneous bolus of saline (10 mL kg-1) and buprenorphine (50 µg kg-1, Vetergesic, UK) before being recovered in 28 ˚C housing. Animals were then transferred to ventilated cages suspended over a heating pad with free access to mashed food and water in normal housing conditions. RNA was isolated using the RNeasy lipid tissue mini kit (Qiagen) as per manufacturer's protocol. RNA-Seq libraries were generated using the TruSeq Stranded mRNA assay (Illumina, Inc.) according to the manufacturer's instructions.