title: GSM6310706: PBN Reaction 4; Mus musculus; RNA-Seq
library_construction_protocol: C57BL/6J mice were transcardially perfused with a cold artificial CSF solution containing NMDG (NMDG-aCSF), modified from (Ting et al., 2018). All steps were performed under continuous oxygenation and CO2 buffering using 95%/5% O2/CO2. Brains were rapidly harvested, and coronal slices (200 µm) spanning the PBN were prepared using a vibrating microtome (VT1200, Leica Biosystems). Slices were allowed to recover in NMDG-aCSF containing 500 nM TTX, 10 µM AP-V, 10 µM DNQX (NMDG-aCSF-R) for 20 minutes and the PBN was subsequently isolated using tissue punches (500-750 μm). The isolated tissue was then enzymatically digested using 1 mg/mL Pronase (Roche) for 50 minutes, and enzymatic digestion was quenched with 0.05% BSA. Cells were then mechanically dissociated using a fire-polished glass pipet with an internal diameter of 200-300 µm, filtered through a 40 μm strainer, washed, and depleted of dead cellular fragments using a commercial kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The remaining cells were resuspended in PBS containing 0.05% BSA at a concentration of 1,000 cells/µL. Single-cell RNA libraries were generated using Chromium Single-Cell 3' v2 chemistry (10X Genomics, Pleasanton, CA) following the standard manufacturer protocol. A pool of ~17,000 cells harvested from 5 mice were loaded per reaction, with the first pool run on a single reaction and the second pool spread across three reactions. For each library, cDNA amplification was performed using 12 cycles and indexing was performed using 11 cycles. Library size distribution and concentration were determined using a Qubit HS DNA Assay (Invitrogen, Waltham, MA) or High Sensitivity D5000 ScreenTape (Agilent Technologies, Santa Clara, CA). Each reaction library was sequenced on two lanes of an Illumina HiSeq 4000 using 2x150 chemistry by Genewiz, Inc (South Plainfield, NJ) following the standard 10X Genomics v2 paired-end configuration. Approximately 875 million reads were generated per lane with a mean sequencing saturation of 82.6%. Sequences were aligned to the mm10-3.0.0 genome, and digital expression matrices were created using 10X Genomics Cell Ranger v3.1.0 with 128 GB of memory on 24 cores.