GSM5229309: Whole Cord WT rep1; Mus musculus; RNA-Seq
The entire developing spinal cord was dissected from one Hb9:GFP embryo from one litter and another Hb9:GFP embryo from another litter in HBSS with HEPES (note: cells were not sorted for GFP). Cell dissociation was conducted using Papain following the manufacturer's instruction (Worthington Biochemical). Specifically, spinal cords were placed in 750 uL papain solution in 37C water bath for 20 minutes, gently triturated, incubated again for additional 25 minutes, fully triturated, and quenched with 250 uL ovomucoid solution. Dissociated cells were then spun down at 150 rcf for 5 minutes. The cell pellet was resuspended in 5% FCS in DMEM/F12 with HEPES without phenol red, and the cells were passed through a cell strainer. Via FACS (FACSDIVA, BD), 20,000 cells were collected (not gated for GFP) into 500 uL of 5% FCS with DMEM/F12 with HEPES without phenol red. Sorted cells were spun down at 300 rcf for 2 minutes, and 370uL were removed to leave remaining 130 uL. The cells were resuspended in this remaining media. 32.5uL of the resuspended cells (~5,000 cells) were loaded into 10X Chromium Controller using Chromium Single Cell 3' v2 reagents. Sequencing library was prepared following the manufacturer's instructions (10X Genomics). 14 cycles were used for cDNA amplification, and 10 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 at the UCSD IGM Genomics Center. Chromium Single Cell 3' v2