GSM7295087: AD graft; clone 2; biol rep1; Homo sapiens; RNA-Seq
Frozen grafts were homogenized using a Dounce homogenizer in a tissue homogenizing buffer that contains 30% sucrose and 0.1% Triton-X 100. 15 strokes with the loss pestle followed by 15 strokes with the tight pestle were applied to each graft. The brain lysate was then mixed well using P1000 pipette followed by filtration through 35 μm nylon mesh (Corning; 352235) followed by 10 min centrifugation of 1000g at 4°C. The pellet was resuspended in homogenizing buffer and filtered through 35 μm nylon mesh again followed by 10 min centrifugation of 1000g at 4°C. The pellet was resuspended in PBS supplemented with 1% BSA and a RNAase inhibitor (Invitrogen; SUPERaseIN; AM2696). Chromium Controller (10x Genomics) was used and single Cell 3' Reagent Kit v2 or v3 (Chromium Single Cell 3' Library & Gel Bead Kit v2, catalog number: 120237; Chromium Single Cell A Chip Kit, 48 runs, catalog number: 120236; 10x Genomics) was used. Illumina NOVAseq 6000 platformV4 with 150bp paired end reads was applied for the sequencing step.