Genomics chromium platform

DNA was extracted from stool samples using a mechanical bead-beating approach with the Mini-Beadbeater-16 (BioSpec Products) and 1-mm diameter zirconia/silica beads (BioSpec Products) followed by the QIAamp Fast DNA Stool Mini Kit (Qiagen) according to manufacturer’s instructions. Bead-beating consisted of 7 rounds of alternating 30 s bead-beating bursts followed by 30 s of cooling on ice. For samples subjected to linked-read sequencing, DNA fragments less than ~2 kb were eliminated with a SPRI bead purification approach⁹² using a custom buffer with minor modifications: beads were added at 0.9×, and eluted DNA was resuspended in 50 μl of water. DNA concentration was quantified using a Qubit fluorometer (Thermo Fisher Scientific). DNA fragment length distributions were quantified using a TapeStation 4200 (Agilent Technologies).
Short-read sequencing libraries were prepared with either the Nextera Flex or Nextera XT kit (Illumina) according to manufacturer’s instructions. Linked-read sequencing libraries were prepared on the 10× Genomics Chromium platform (10× Genomics). Linked-read libraries have a single sample index, and were pooled to minimize the possibility of barcode swapping between samples from patients who were roommates (see Supplementary Methods). Libraries were sequenced on an Illumina HiSeq 4000 (Illumina).

For the Mouse-Wang-2021 dataset, single live nonCM cells were isolated from the left and right ventricles of adult mice and transcriptionally profiled using the 10× Genomics Chromium platform (10× Genomics, Inc., Pleasanton, CA, USA). Low-quality cells expressing <200 genes and genes expressed in less than three cells were first filtered out from each of the two biological replicates. For each sample, the top 2000 HVGs were identified. Then, the two biological replicates were merged and corrected for batch effect using Seurat v3 [28 (link)]. Data dimensions were reduced by PCA, and unsupervised clustering was performed to identify cell clusters using the FindClusters function, with the first 20 PCs and the resolution parameters set to 0.5. The obtained clusters were defined according to the expression levels of known cell-type-specific markers. Since we only focused on nonCMs, cluster(s) highly expressing canonical markers of cardiomyocytes were removed from downstream analysis.

As previously described by Huuki-Myers et al. 2023 (46 ), paired (meaning both types of -omics were measured on the same N=19 tissue blocks) snRNA-seq measured on the 10x Genomics Chromium platform and spatially-resolved transcriptomics data measured on the 10x Genomics Visium platform were generated from tissue blocks collected from 3 different positions across the anterior-posterior axis of the DLPFC (designated posterior, anterior, and middle) from 10 adult neurotypical control donors (Table S1). In our analyses here, we only use the snRNA-seq libraries to generate the pseudobulk RNA-seq profiles. In addition to the snRNA-seq libraries generated from these tissue blocks, there was also paired bulk RNA-sequencing, and single molecule fluorescent in situ hybridization (smFISH)/immunofluorescence (IF) using RNAScope/IF technology generated and described in Huuki-Myers et al. 2024 (26 ) measured in the same tissue blocks. The smFISH/IF data was used to measure the cell type composition in the same tissue samples serving as a “gold standard” to compare the estimated cell composition in the bulk RNA-seq predicted by the deconvolution algorithms in (26 ). The fact that all three (snRNA-seq, bulk RNA-seq, and smFISH/IF) technologies were measured on the same tissue blocks helps to minimize potential donor-specific unwanted variation or batch effects.

Adult mice were sacrificed and inner ears were dissected. The lateral wall of the cochlea was microdissected from the bony wall of the cochlea. Localizing the pigmented strip in the cochlear lateral wall, the SV was microdissected from the spiral ligament using fine forceps. Microdissection of the SV from 2 cochlea were accomplished in less than 4 min. Multiple lab personnel experienced with the microdissections were utilized to minimize dissection time. Samples were collected at the same time of day across individual mice and batches. For each collection, less than 1 h was spent prior to single cell or single nucleus capture on the 10x Genomics Chromium platform. A total of 10 mice and 12 mice were used for single cell and single nucleus RNA-seq experiments, respectively.