Unknown,Transcriptomics,Genomics,Proteomics

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Single cell RNA seq of Cell Multiplexing Oligo, pooled Pleurodeles waltl and Notophthalmus viridescens spleens

ABSTRACT: The goal of this study was to test if lipid-based cell hashing worked in salamander species and to test how lipid-based hashing compared to demultiplexing samples based on species origin and single nucleotide polymorphisms (SNP). Spleens were taken from four animals in total: one adult Pleurodeles waltl (female, 23.5grams and 16.1cm snout-to-tail, strain: tgSceI(CAG:loxP-GFP-loxP-Cherry)Simon), one adult Pleurodeles waltl (male, 13.95g and 15.7cm, tgSceI(CAG:loxP-GFP-loxP-Cherry)Simon), one adult Notophthalmus (female, 4.45g and 10.6cm, WT), and one adult Notophthalmus (male, 3.55g and 10.2cm, WT). Samples were stained with CM304 (Pleurodeles female), CMO305 (Pleurodeles male), and CMO306 (pool of both Notophthalmus samples) as per 10x Genomics 3’ Cellplex labeling protocol (Demonstrated Protocol, CG000391). These samples were processed through the 10x Controller and with Cellranger 7.0 using a dual species reference. The sample origin was determined by lipid-based hashing and then compared to mapping rates to each species transcriptome and using SNP-based demultiplexing.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Notophthalmus viridescens

SUBMITTER: Nicholas Leigh

PROVIDER: E-MTAB-12182 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Single cell RNA sequencing (scRNAseq) has emerged as an essential technique in biology. Given the complexity and cost of experiments it is critical that they are well planned and executed. An essential component to scRNAseq is the ability to have biological replicates. This adds to the potential cost and complexity of the experiment, but is essential as it has been shown that false discoveries are possible when lacking information pertaining to cell origin. One option to overcome the financial and experimental constraints of biological replicates in scRNAseq data is to pool samples. Upon pooling it is essential to then understand the sample origin of each cell. Experiments in humans have shown that when pooling multiple genetically distinct individuals into one sample the genetic diversity (i.e., single nucleotide polymorphisms (SNP)) can be used to assign cells to their sample origin. This approach is called SNP-based demultiplexing. The question of whether lab species, like Pleurodeles waltl, and various other non-traditional model species harbor enough genetic diversity to enable such approaches is unclear. To formally test the whether SNP-based demultiplexing is possible across various species we designed this experiment in which three spleens from three different Pleurodeles waltl from animals expressing unique fluorescent genes (one female tgTol2(CAG:Nucbow CAG:Cytbow)Simon (5.67g weight, 10.8cm snout-to-tail length), one male tgSceI(CAG:loxP-GFP-loxP-Cherry)Simon (5.36g, 11.1cm), and one female tgTol2(CAG:loxP-Cherry-loxP-H2B::YFP)Simon (6.25g, 10.8cm)) were collected. Spleens were filtered through 70um nylon filters followed by FACS, gating on fluorescent positive cells and excluding erythrocytes. After FACS cells were pooled into one tube and subjected to 10x Genomics 3' v3.1 scRNAseq aiming for 10,000 cells. Spleens were filtered through 70um nylon filters followed by FACS gating on fluorescent positive cells and excluding erythrocytes. Samples were prepared and sequenced according to 10x Genomics recommendations. After sequencing, using reads mapping to fluorescent genes to identify animal origin we then compared the ability of SNP-based demultiplexing to properly assign cells to the correct animal.

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Single cell RNA seq of Cell Multiplexing Oligo, pooled Pleurodeles waltl and Notophthalmus viridescens spleens

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