title: GSM6133160: adult_mouse_hippocampus; Mus musculus; RNA-Seq
library_construction_protocol: Animals were perfused with PBS before the brain extraction. Cells were isolated using the Adult Brain Dissociation Kit (ABDK, Miltenyi Biotec). In brief, mice were anaesthetised (Narcorene, 100mg/kg, i.p., Merial GmbH, Germany) and perfused transcardially with 10 ml of 0.9 % saline solution containing 1 U/ml of heparin (Heparin-Natrium-250 000-ratiopharm, Ratiopharm, Ulm, Germany) followed by 50 ml of 0.1M PBS (pH 7.4). Next, the brains were gently extracted from the skull (Papouin 2018) and immediately placed in ice cold HBSS. For the hippocampus isolation, brains where put on an ice cold metal platform and the hippocampus was dissected out of the whole brain as described by Spijker. Hippocampi were then placed in the ABDK enzyme mix and dissociated for 30 min at 37°C according to the manufacturer's instructions in the presence of 10mM 4sU. Afterwards the cell suspension underwent myelin removal with 35% SIP (Solution Isotonic Percoll) for 25min at 4°C. Live cells were sorted next with all steps being performed on ice and/or with pre-chilled buffers and equipment at 4°C. Cells were stained with BD Pharmingen™ Calcein AM dye for live/dead cell staining at 10 μM final concentration for 30-40min on ice. Afterwards the cell suspension was spun down for 5 min at 300g, before being resuspended in 500 μl of FACS buffer. Next, cells were sorted using an ARIA II sorter with 70 µl nozzle at the speed of 10k cells/sec. Cells were spun down and resuspended in HBSS, before cold methanol was added drop-wise while gently agitating on a vortexer. Afterwards the cells were fixed at -20°C for at least 30 minutes. For IAA treatment the samples were brought back to RT and IAA dissolved in 20% HBSS / 80% MeOH (Th. Geyer #1437.2511) was added to a final concentration of 10 mM IAA. After converting for 13 hours, the cells were spun down at 1000 g and 4°C for 10 min, the supernatant was removed and quencing buffer added (3x SSC (Carl Roth GmbH #1054.1), 0.1% BSA,  1 U/µl SuperRNase IN (Life Technologies GmbH #AM2694), 100 mM DTT. We incubated the sample at RT for 5 min on order to quench the remaining IAA. Afterwards the cells were spun down again and resuspended in a small amount of resuspension buffer (1.5x SSC,  0.5 U/µl SuperRNase IN,  0.01% BSA) and loaded onto the chromium controller using a Chromium Single Cell 3' v3.1 kit (10x Genomics #1000121). Importantly, the volume of SSC buffer introduced into reverse transcription needs to be as low as possible to avoid inhibiting the reaction.After successful encapsulation we proceeded with library preparation according to the manufacturer's instructions.