GSM5199029: B8B-GEX: MIBC_rxn8B; Homo sapiens; RNA-Seq
Nuclei were isolated from fresh frozen muscle-invasive bladder cancer (MIBC) tumors using a method modified from a recent single-nuclei RNA-sequencing (snSeq) study41. The ST-SB buffer from that study was modified by removing Tween-20 and supplementing with 0.04U/L Protector RNase Inhibitor (Roche). Unless otherwise specified, all sample manipulation was performed on wet ice with wide-bore pipet tips (Rainin) and all centrifugations were performed with a swinging bucket rotor maintained at 4C for 5 minutes at 850x g. In brief, the frozen tissue was transferred onto a plate on dry ice and crushed into ≤ 1mm3 pieces. This was then transferred to a 2mL dounce homogenizer (Kimble, cat: 885300-0002) on wet ice containing 1mL of Nuclei EZ lysis buffer (Sigma, cat: NUC101). The tissue was then dounced approximately 20x with Pestle A followed by 20x with Pestle B. The lysis was then quenched by adding 1mL of ST-SB. The sample was filtered through a pre-wetted 30m filter (Miltenyi Biotec, cat: 130-041-407) into a 15mL conical tube. The homogenizer was rinsed 3x with 1mL of ST-SB and this was transferred through the same 30m filter into the 15mL conical tube. The sample was then centrifuged, the resulting supernatant removed, and the pellet resuspended with 500L of ST-SB. The sample was then passed through a pre-wetted 20m filter (Miltenyi Biotec, cat: 130-101-812) into a 1.5mL protein lo-bind microcentrifuge tube (Eppendorf, cat: 022431081) and centrifuged. At this point, Totalseq hashing antibodies (Biolegend, clone Mab414) were also centrifuged at 14,000xg for 10 minutes at 4C. The sample pellet was then resuspended in 100L of ST-SB and 10L of Human TruStain FcX block (Biolegend, cat: 422301) was added. The sample was pipet mixed and incubated at 4C for 5 minutes. Then 1.5g of the appropriate hashing antibody was added to the appropriate samples, pipet mixed, and incubated at 4C for 15 minutes. The samples were pipet mixed once halfway through this incubation. The samples were then washed 2x with 1mL of ST-SB, pooled appropriately, and filtered through another 30m and 20m filter. Nuclei concentration was quantified by mixing an aliquot of the sample with DAPI at a final concentration of 0.025 mg/mL in H2O. Samples were finally processed according to 10x Genomics protocol for the 3' v3.1 assay and were super-loaded to target of 20,000 nuclei recovery. We observed that nuclei yield less total cDNA than cells, therefore we increased the first cDNA amplification cycle number by 2. Hashing libraries were generated according to the Biolegend Totalseq protocol for the 3' v3.1 assay. In total, 57 samples from 25 patients were processed. All samples were sequenced by the Cedars-Sinai Applied Genomics, Computation & Translational Core on a Novaseq to a sequencing saturation of approximately 60%.