title: GSM7211316: Microglia, Bile_duct_ligation, 14days, rep2; Mus musculus; RNA-Seq
library_construction_protocol: After euthanasia using an overdose of ketamine (87.5 mg/kg) and xylazine (12.5 mg/kg), mice were perfused transcardially using 10 ml 0.2 % heparin in ice-cold D‐PBS per mouse  at a flow rate of 4.50 ml/min. Whole brain samples were isolated from the skull, weighed and collected in 1.5 mL ice-cold 1x HBSS -/- . Brains were cut into pieces using spring scissors and slurry was dissociated into single cell suspensions using the Neural Tissue Dissociation Kit (P) (Miltenyi Biotec). Cells were enzymatically dissociated using activated enzyme (P) for 15 min at 37 °C and enzyme (A) for 2x10 min at 37 °C under continuous nutation, with mechanical dissociation by trituration 10 times with a 5 ml serological pipet in between enzymatic dissociation steps. Subsequently, samples were passed through a 70 µM cell strainer. Myelin was removed by centrifugation (300g, 11 min, room temperature, low acceleration/break) of samples in 24 % Percoll PLUS and 75 U/ml DNase. After aspiration of the myelin layer and supernatant, cell pellet was resuspended in 50 µL of FACS buffer (0.5% BSA in D‐PBS). This process was repeated twice. Single cell suspensions were pre-incubated with Fc Block (1/100; BD Biosciences, 553142) for 10 min at 4 °C and stained with appropriate antibodies diluted at 4 °C in the dark for 30 min. After washing and centrifugation (400g, 7min, 4°C), supernatans was removed and pellets were resuspended in FACS buffer and transferred through a 35 µm mesh into a 5 ml Falco®Round‐Bottom Polystyrene Test Tube with Cell Strainer Snap Cap. Cell viability was assessed using DAPI 1/200, added immediately prior to sorting. Cell sorting was performed on the FACSymphony S6 using the 85 µm nozzle.  Cells were sorted into 2 ml Eppendorf tubes containing 450 µL RLT Plus lysis buffer (QIAGEN) containing 1% β-mercaptoethanol. The samples were extensively vortexed and stored at ‐80 °C until RNA isolation. Total RNA from sorted cells was extracted using the RNeasy ®Plus Micro Kit (QIAGEN, 74034) according to the manufacturer's instructions. The concentration and purity of the RNA was determined using the Agilent RNA 6000 Pico Kit (Agilent) and the Agilent 2100 Bio-Analyzer. A RIN value of 7 or higher was required to proceed. 1 ng of total RNA was used for constructing sequencing libraries. RNA libraries were prepared for sequencing using standard Illumina protocols.