Illumina NovaSeq 6000 paired end sequencing; RNA-seq of ex vivo primate cells
RNA-seq of ex vivo primate cells
The brain tissue was removed and collected from a four-year-old monkey in accordance with Regulations for the Administration of Affairs Concerning Experimental Animals. The two hippocampi were quickly dissected into a 10-cm dish containing 10 ml pre-cold Hank's Balanced Salt Solution (HBSS) (Hyclone, Logan, UT) using a sterile scalpel and surgical scissors.  The dissociation solution including 40 U/mL papain (Worthington), 20 mg/mL Collagenase type II(Worthington), 0.25% trypsin-EDTA (Gibco) and 5 mg/mL DNase I (Roche) in ACSF warmed at 34°C for 10min before use. The tissues were transferred to dissociation solution and incubated for approximately 60 min at 34°C. Crucially, dissociation solution was always bubbled with a mixture of 95% O2 and 5% CO2 during the enzymatic digestion. After incubation, the tube was briefly spun at 500 rpm, and the supernatant was gently removed. The pieces were resuspended with 10% fetal bovine serum in ACSF to stop digestion, followed by manual trituration with fire-polished pipettes, and filtering through a 70 um ACSF-equilibrated cell strainer. The dissociated cells were removed debris and dead cells using Myelin Removal Beads II (Miltenyi) and Dead Cell Removal Kit (Miltenyi) according to manufacturer's instructions, and then washed 2 times with 0.04% PBS-BSA, gently resuspended in 0.04% PBS-BSA. Cells were counted using an automated cell counter (Bio-Rad, TC20) after visual inspection. Using the above optimized dissociation protocol, we obtained high quality cell suspensions (suspension volume and cell density?, rate of survival > 90%).  10X Genomics standard protocols