GSM3559992: PFC P21 scRNA-Seq Sample3; Mus musculus; RNA-Seq
For single cell dissociation of Prefrontal cortex (PFC), the mice were anesthetized and the entire brain was quickly removed and transferred into ice-cold Hibernate A/B27 medium (60 ml Hibernate A medium with 1 ml B27 and 0.15 ml Glutamax). The coronal sections containing PFC were cut with brain matrix and further sliced into 0.5 mm slices in ice-cold Hibernate A/B27 medium. Then the PFC tissue was dissected from each slice under dissection microscope and subjected to tissue dissociation (Figure S1A). The NAc tissues were further dissociated into single-cell suspension with a papain-based dissociation protocol (Brewer and Torricelli, 2007) with some modifications. Briefly, the tissues from two animals were cut into small pieces and incubated in dissociation medium (Hibernate A-Ca medium with 2 mg/ml papain and 2X Glutamax) at 300C for 35-40 min with constant agitation. After washing with 5 ml Hibernate A/B27 medium, the tissues were triturated with fire polished glass Pasteur pipettes for 10 times in 2 ml Hibernate A/B27 medium to generate single-cell suspension, which was repeated for three times. To remove debris, the 6 ml single-cell suspension was loaded on a 4-layer OptiPrep gradient (Brewer and Torricelli, 2007) and centrifuged at 800 g for 15 min at 40C. Fractions 2 – 4 were then collected and washed with 5 ml Hibernate A/B27 medium and 5 ml DPBS with 0.01% BSA. The cells were spun down at 200 g for 3 min and re-suspended in 0.2 ml DPBS with 0.01% BSA. A 10 µl cell suspension was stained with Trypan Blue and the live cells were counted. During the entire procedure, the tissues or cells were kept in ice-cold solutions except for the papain digestion. The cells suspension was diluted with DPBS containing 0.01% BSA to 300 - 330 cells/µl for single cell capture. Single cells and barcoded beads were captured into droplets with 10X Chromium platform (10X Genomics) according to the protocol from the manufacturer (Zheng et al., 2017). After cell capture, reverse transcription, cDNA amplification and sequencing library preparation were perform as described in the same protocol (Zheng et al., 2017). The libraries were sequenced on Illumina Hiseq 2500 sequencer with pair-end sequencing (Read1: 26bp, Index: 8bp, Read2: 98bp).