GSM7646153: GEX_43R; Homo sapiens; RNA-Seq
To isolate nuclei, the frozen tissue (50-60 mg) was thawed for 2 minutes in 2 mL of ice-cold Nuclei EZ Lysis Buffer (Sigma, Cat # NUC-101), supplemented with EDTA-free protease inhibitors (Sigma, Cat # 11836170001) and 0.4 U μL−1 recombinant RNase inhibitors (Takara Bio USA, Cat # 2313A) before homogenizing with a glass Dounce tissue grinder (10-15 strokes with pestle A, followed by 10 strokes with pestle B. Sigma, Cat # D8938). An additional 2 mL of lysis buffer supplemented with protease and RNase inhibitor was added to the homogenized tissue and incubated for 5 minutes. The nuclei suspension was then filtered through a 40-μm cell strainer (Corning, Cat # 352340), followed by centrifugation at 300 x g for 5 minutes. The nuclei pellet was resuspended with 900 μL debris removal solution (Miltenyi Biotech, Cat # 130-109-398) containing 0.4 U μL−1 RNase inhibitors and 3.1 mL of Dulbecco's phosphate-buffered saline (DPBS) without calcium and magnesium. Next, 4 mL of DPBS was overlaid carefully onto the top of the nuclei resuspension. The nuclei were then pelleted again by centrifugation for 5 minutes at 3,000 x g. After removing the supernatant, the nuclei were resuspended in 500 µL of 1% BSA in DPBS with 0.4 U μL−1 RNase inhibitors. All procedures for nuclei isolation were carried out on ice or at 4°C. The density of the nuclei suspension was determined using a C-chip-disposable hemocytometer (INCYTO, Cat # DHC-B02-5) with trypan blue staining (Bio-Rad, Cat # 1450013). The 59 samples were processed in 10 batches. Each batch had 5 to 6 samples from individuals of the same sex randomly selected from each of the AD and APOE genotype groups, i.e., control APOE2 carriers, control APOE3 homozygotes, control APOE4 carriers, AD APOE2 carriers, AD APOE3 homozygotes, and AD APOE4 carriers (6 groups in total). The samples from males and females were processed in alternating batches. A total of 5000 nuclei per sample were loaded to the Chromium microfluidic chip for cDNA library preparation using the Chromium Single Cell 3' Library and Gel Bead kit v3 (10X Genomics, Cat # 1000075) following the manufacturer's instructions.