GSM5229308: Lumbar neurons 1; Mus musculus; RNA-Seq
Lumbar segments were dissected from Vglut2; tdTomato (2 female and 1 male) and Vgat; tdTomato (2 female and 1 male) in aCSF. Cell dissociation was conducted using Papain following the manufacturer's instruction (Worthington Biochemical) with 5% Trehalose, 50uM APV, and 800uM KA added during the dissociation process (41). Via FACS (FACSDIVA, BD), 30,000 Vglut2; tdTomato cells and 30,000 Vgat;tdTomato cells were sequentially collected into 500ul of FACS buffer (DMEM w/o Phenol Red; 1mM EDTA; 25mM Hepes pH7.0; 5% FBS). Cell suspension was centrifuged at 300 rcf for 2 minutes, and supernatant was removed leaving 60 ul of FACS buffer and cell pellet. The cells were resuspended, and 33.4 ul of the resuspended cells (~30,000 cells) were loaded onto the 10X Chromium Controller using Chromium Single Cell 3' v2 reagents. Sequencing library was prepared following the manufacturer's instructions (10X Genomics). 9 cycles were used for cDNA amplification, and 12 cycles were used for library amplification. The resulting sequencing library was sequenced with Paired End reads, with a Read 1 of 26 basepairs and a Read 2 of 98 basepairs, on an Illumina HiSeq 4000 and demultiplexed using bcl2fastq v2.18.0.12 at the UCSD IGM Genomics Center. Chromium Single Cell 3' v2