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. 2022 Jun 7;3(2):101446.
doi: 10.1016/j.xpro.2022.101446. eCollection 2022 Jun 17.

Multi-omics profiling of single nuclei from frozen archived postmortem human pituitary tissue

Natalia Mendelev  1 , Michel Zamojski  1 , Mary Anne S Amper  1 , Wan Sze Cheng  1 , Hanna Pincas  1 , Venugopalan D Nair  1 , Elena Zaslavsky  1 , Stuart C Sealfon  1 , Frederique Ruf-Zamojski  1
Affiliations

Multi-omics profiling of single nuclei from frozen archived postmortem human pituitary tissue

Natalia Mendelev et al. STAR Protoc. .

Abstract

Concomitant profiling of transcriptome and chromatin accessibility in isolated nuclei can reveal gene regulatory control mechanisms in health and disease. We report a single nucleus multi-omics analysis protocol optimized for frozen archived postmortem human pituitaries that is also effective for frozen ovine and murine pituitaries and human skeletal muscle biopsies. Its main advantages are that (1) it is not limited to fresh tissue, (2) it avoids tissue dissociation-induced transcriptional changes, and (3) it includes a novel, automated quality control pipeline. For complete details on the use and execution of this protocol, please refer to Ruf-Zamojski et al. (2021) and Zhang et al. (2022).

Keywords: Cell Biology; Genomics; Health Sciences; RNAseq; Sequencing; Single Cell.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Flowchart of the protocol The key steps of the protocol from nuclei isolation to data analysis are depicted.
Figure 2
Figure 2
Purification of nuclei with gradient centrifugation Following gradient centrifugation, nuclei are at the border between the 30% and 35% OptiPrep. The suspension around this band is taken for further cleanup of the nuclei in preparation for downstream sn assays.
Figure 3
Figure 3
Quality assessment of nuclei preparation Following nuclei isolation and cleanup steps, nuclei quality is assessed and nuclei are counted on a Nexcelom fluorescent cell counter. (A) Image showing highly concentrated nuclei and clumps in the preparation. (B) Image showing the same preparation after dilution and pipetting to remove large clumps.
Figure 4
Figure 4
Representative Bioanalyzer traces for snRNAseq QC (A–C) Shown are representative traces of successful Bioanalyzer QC analyses for snRNAseq (A) and sn multiome (B), each at the cDNA amplification step (i) and following library preparation (ii). Failed QC traces are shown in (C).
Figure 5
Figure 5
Representative Bioanalyzer traces for snATACseq QC (A–C) Shown are representative traces of successful Bioanalyzer QC analyses for snATACseq (A) and sn multiome (B) libraries. Failed QC traces are shown in (C).
Figure 6
Figure 6
Representative Bioanalyzer traces for library pool QC (A and B) Shown are representative traces of successful Bioanalyzer QC analyses after the pooling of snRNAseq (A) or snATACseq (B) libraries. Twenty-four libraries were pooled in both (A and B).
Figure 7
Figure 7
Cluster identification and quality assessment Results obtained for the frozen archived human pediatric pituitaries processed for sn multiome. (A) Preliminary UMAP including multiplet and debris/dead cell clusters. (B) UMI/RNA count. (C) Mitochondrial gene content. (D) Clean UMAP following the removal of multiplet and debris/dead clusters.
See this image and copyright information in PMC

References

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