Received: JanuAccepted: ApPublished: September 7, 2023Ĭopyright: © 2023 Ruiz Daniels et al. PLoS ONE 18(9):Įditor: Sven Winter, University of Veterinary Medicine Vienna: Veterinarmedizinische Universitat Wien, AUSTRIA (2023) A versatile nuclei extraction protocol for single nucleus sequencing in non-model species–Optimization in various Atlantic salmon tissues. h5 files with corresponding cell metadata as csv file.Citation: Ruiz Daniels R, Taylor RS, Dobie R, Salisbury S, Furniss JJ, Clark E, et al. Filtered bc matrix file from aggregated cellRanger analysis is available for snATAC and snRNA-seq as. Supplementary_files_format_and_content: Bulk tissue TPM data is save as R data file (rda). ‘Pseudo-bulk’ chromatin accessibility coverage profiles of gene body and upstream promoter regions were visualized using the Signac (v0.2.0) function CoveragePlot, while gene expression signals were visualized using Seurat (v3.1.2) snRNA-seq cell type assignments

We used linear regression model, similar to our previous work, to remove the effects of biological covariates Reads were aligned to the reference genome (GRCh38.p13) using STAR aligner (v2.6.1) and aligned BAM file QC metrics were obtained using PicardTools (v2.21.3)īulk RNA-seq gene expression was quantified as transcripts per million (TPM) using Salmon (v1.3.0). We constructed a merged gene by barcodes matrix for all snRNA-seq libraries using the cellranger aggr function, normalizing for read depth across libraries.įor bulk RNA-seq, we performed QC on raw fastq reads, sequenced at median read-depth of 50-million paired-end reads/sample, using FastQC. Gene expression was quantified by mapping reads from snRNA-seq libraries to a pre-mRNA build of the same reference genome, ensuring that we account for unprocessed nuclear transcripts, using cellranger count (v3.0.2 10X Genomics). Libraries were prepared using 10x genomics Chromium Single Cell ATAC platform (V1), 10x genomics chromium V3 for snRNA-seq and SMARTer Stranded Total RNA Sample Prep kit-HI Mammalian for bulk RNA-seqĬhromatin accessibility was quantified in single nuclei by mapping snATAC-seq reads to the hg38 reference genome and calling peaks using cellranger-atac count (v1.1.0, 10X Genomics) We constructed a merged peak by barcodes matrix for all snATAC-seq libraries using the cellranger-atac aggr function, which normalizes accessibility signals by sampling fragments from GEM wells to ensure uniform distributions of enriched Tn5 cut sites across libraries. For bulk RNA-seq, total RNA was isolated using Mini Nucleospin RNA kit, Takarabio Libraries were prepared using 10x genomics Chromium Single Cell RNA-seq platform (V3)įor snRNA-seq and snATAC-seq, single nucleus suspensions were isolated from about 50mg frozen human prefrontal cortex following 10x genomics recommendation. Single nucleus suspensions were isolated from about 50mg frozen human prefrontal cortex, homogenized in Nuclei EZ Lysis buffer (Sigma). Isolation: unbiased total nuclei isolation GEO help: Mouse over screen elements for information.

10x chromium v3 buffers