GSM8774894: CX3_VE_BS; Mus musculus; RNA-Seq
All samples were handled and processed under Air Clean PCR Workstation. Frozen brain tissue was dissociated using a Dounce homogenizer with 1X lysis buffer (Nuclei PURE Lysis Buffer, Sigma L9286). Nuclei suspension is filtered using a 35 um Cell Strainer and centrifuged at 600g for 5 min at 4 ºC. Pellet was resuspended in wash buffer [1X SCC (Invitrogen, cat no AM9770)  20 mM DTT, 1% BSA and RNAse inhibitor (Ambion, cat no AM2682)] and centrifuged at 600g for 5 min at 4 ºC. Nuclei were stained with DAPI at 1 mg/1 mL (Invitrogen, cat no D1306 and DAPI+ nuclei were FACS-sorted with a BD FACS Aria with a 100-μm nozzle and a sheath pressure 20 psi. Sorted nuclei were centrifuged in 5mL tubes in a swinging bucket centrifuge at 600g for 5min. RIN was determined using BioAnalyzer. The single-nuclei RNA-Seq of FACS-sorted nuclei suspensions was performed on Chromium instrument (10X genomics) following the user guide manual for 3′ v3.1. In brief, FACS-sorted cells were washed once with PBS containing 1% bovine serum albumin (BSA) and resuspended in PBS containing 1% BSA to a final concentration of 700–1,300 cells per μl. The viability of cells was above 80%, as confirmed with 0.2% (w/v) Trypan Blue staining (Countess II). Cells were captured in droplets. Following reverse transcription and cell barcoding in droplets, emulsions were broken and cDNA purified using Dynabeads MyOne SILANE followed by PCR amplification per manual instruction. Samples were multiplexed together on one lane of 10X Chromium (using Hash Tag Oligonucleotides - HTO) following previously published protocol 67. Final libraries were sequenced on Illumina NovaSeq S4 platform (R1 – 28 cycles, i7 – 8 cycles, R2 – 90 cycles). The cell-gene count matrix was constructed using the Sequence Quality Control (SEQC) package 68. Viable cells were identified on the basis of library size and complexity, whereas cells with >20% of transcripts derived from mitochondria were excluded from further analysis.