Novaseq 6000 with s4 flow cell

Single-nucleus suspensions were counted using disposable counting chambers (Bulldog Bio, Portsmouth, NH) on a Leica DMi 1 microscope by a second investigator not involved in tissue processing. A total of 15,000–20,000 nuclei were loaded per channel on a Chromium controller using Chromium Next GEM Single Cell 3ʹ v3.1 reagents (10X Genomics, Pleasanton, CA) placed inside the bio-safety cabinet, and single-nucleus RNA-seq libraries were prepared per the manufacturer’s instructions (increasing the recommended initial cDNA amplification cycles by one to account for lower amounts of RNA from nuclei compared to whole cells). Single-nucleus RNA libraries were analysed and quantified using TapeStation D1000 screening tapes (Agilent, Santa Clara, CA) and Qubit HS DNA quantification kit (Thermo Fisher Scientific). Libraries were pooled equimolarly and quantified using quantitative PCR. Libraries were sequenced on a NovaSeq 6000 with S4 flow cell (Illumina, San Diego, CA) using paired-end, single-index sequencing with 28 cycles for read 1, 8 cycles for i7 index, and 91 cycles for read 2.

Gene expression libraries were quantified as described above and equimolarly mixed before sequencing. Libraries were sequenced targeting a minimum of 2e⁸ reads per sample using paired-end sequencing with 2x150 bp on a NovaSeq 6000 with S4 flow cell (Illumina, San Diego, CA).