Single-Nucleus RNA Sequencing of Frozen Tissue

A pilot single-nucleus RNA sequencing experiment was undertaken to compare single-cell versus single-nuclear results from a matched sample. The biopsy from a 58-year-old male was collected fresh and divided into 8 segments, evenly distributed to be processed fresh for single cell RNA sequencing as above, and the remainder was flash frozen in liquid nitrogen. The sample was later retrieved from liquid nitrogen and processed on dry ice according to the protocol in^{88 (link)} with a lysis buffer containing: 0.32 mM sucrose (BioShop SUC507.1), 5 mM CaCl2 (VWR, 97062-820), 3 mM MgCl2 (Thermo Fisher AM9530G), 20 mM Tris-HCl pH 7.5 (Thermo Fisher, 15567027), 0.1% TritonX-100 (Sigma Aldrich T8787-50ML), 0.1 mM EDTA pH 8.0 (Thermo Fisher AM9260G), 40 U/ml Protector RNAse inhibitor (Sigma Aldrich 3335399001) in UltraPure DNAse/RNAse-free water (Thermo Fisher 10977015). The nuclei were captured and sequenced using 10X Genomics Single Cell 3’ v3 Reagents as above.

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McEvoy C.M., Murphy J.M., Zhang L., Clotet-Freixas S., Mathews J.A., An J., Karimzadeh M., Pouyabahar D., Su S., Zaslaver O., Röst H., Arambewela R., Liu L.Y., Zhang S., Lawson K.A., Finelli A., Wang B., MacParland S.A., Bader G.D., Konvalinka A, & Crome S.Q. (2022). Single-cell profiling of healthy human kidney reveals features of sex-based transcriptional programs and tissue-specific immunity. Nature Communications, 13, 7634.

Publication 2022

SUC507.1 AM9530G T8787-50ML AM9260G Protector RNAse inhibitor UltraPure DNAse/RNAse-free water

Biopsy Buffer Cell Dnase Dry ice Edta Frozen Male Mgcl2 Nitrogen Nuclei Rnase Single nucleus Sucrose Tris

Corresponding Organization :

Other organizations : University Health Network, Toronto General Hospital, University of Toronto, Princess Margaret Cancer Centre

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Variable analysis

independent variables

Single-cell versus single-nuclear processing

dependent variables

Transcriptomic profile from the biopsy sample

control variables

Biopsy sample from a 58-year-old male
Sample divided into 8 segments, with 4 segments processed fresh for single-cell RNA sequencing and the remaining 4 segments flash frozen in liquid nitrogen
Frozen samples later retrieved and processed on dry ice according to the protocol in ref. 88
Lysis buffer composition: 0.32 mM sucrose, 5 mM CaCl2, 3 mM MgCl2, 20 mM Tris-HCl pH 7.5, 0.1% TritonX-100, 0.1 mM EDTA pH 8.0, 40 U/ml Protector RNAse inhibitor in UltraPure DNAse/RNAse-free water
Nuclei captured and sequenced using 10X Genomics Single Cell 3' v3 Reagents

controls

Positive control: None specified
Negative control: None specified

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