title: GSM4462799: OB_121316_run2; Mus musculus; RNA-Seq
library_construction_protocol: Dissected olfactory bulbs (including the accessory olfactory bulb and fractions of the anterior olfactory nucleus) were dissociated in 750 µl of dissociation media (DM: HBSS containing 10mM HEPES, 1 mM MgCl2, 33 mM D-glucose) with 28 U/mL Papain and 386 U/mL DNase I (Worthington). Minced tissue pieces were transferred to a 5 mL round-bottom tube (BD). DM was added to a final volume of 3.3 mL and the tissue was mechanically triturated 5 times with a P1000 pipette tip. After 1-hour incubation with rocking at 37°C, the suspension was triturated with a 10 mL pipette 10 times and 2.3 mL was passed through 40 µm cell strainer (BD). The suspension was then mechanically triturated with a P1000 pipette tip 10 times and 800 µL were filtered on the same strainer. The cell suspension was further triturated with a P200 pipette tip 10 times and filtered. 1 mL of Quench buffer (22 mL of DM, 2.5 mL of protease inhibitor prepared by resuspending 1 vial of protease inhibitor with 32 mL of DM, and 2000U of DNase I) was added to the suspension and centrifuged at 300g for 5 min. Cells were resuspended with 3 mL of Quench buffer and overlaid gently on top of 5 mL of protease inhibitor, then spun down at 70g for 10min. The pellet was resuspended using DM supplemented with 0.04 % BSA, and spun down at 300g for 5 min. Cells were suspended in 400 µL of DM with 0.04 % BSA. Drop-seq experiments (cell capture, reverse transcription, PCR, tagmentation), were performed as described in Macosko et al. 2015. Microfluidics devices were obtained from FlowJEM and barcode beads were obtained from Chemgenes. 4 replicates of Drop-seq samples were pooled and sequenced across 3 runs on an Illumina NextSeq 500 platform
attributes.tag: GEO Accession
attributes.value: GSM4462799