GSM9455143: Mouse brain, Rc5d, rep1; Mus musculus; RNA-Seq
The microdissected amygdala tissue pieces were transferred into 1 mL of papain digest solution (in aCSF, supplemented with 5% DNase). The tissue was digested for a total of approximately 40 min at 37 ℃. Following digestion, the tissue was sequentially triturated using large- and small-diameter fire-polished glass pipettes to generate a cell suspension. All collected cell suspension was pooled, filtered through an aCSF-equilibrated 35 μm cell strainer. The filtered cell suspension was pelleted by centrifugation at 300 g for 5 min at 4 ℃. The cell pellet was resuspended in 200 μL of aCSF supplemented with 2.5% DNase. For debris removal, the suspension was layered on top of 1 mL of 5% OptiPrep in aCSF, and centrifuged at 150 g for 10 min at 4 ℃. The final cell pellet was resuspended in 60 μL of aCSF. The cells that have passed the test are washed and resuspended to prepare a suitable cell concentration of 700~1200 cells/ul for 10x Genomics ChromiumTM. The system is operated on the machine. GEMs (Gel Bead in Emulsion) were constructed for single cell separation according to the number of cells to be harvested. After GEMs were normally formed, GEMs were collected for reverse transcription in a PCR machine for labeling. The GEMs were oil-treated, and the amplified cDNA was purified by magnetic beads, and then subjected to cDNA amplification and quality inspection. The Gene Expression Library was constructed with the quality-qualified cDNA. After fragmentation,  adaptor ligation, sample index PCR, etc., the library is finally quantitatively examined. Sequencing: The final library pool was sequenced on the Illumina Nova 6000 instrument using 150-base-pair paired-end reads.