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

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).

SUBMITTER: Mendelev N

PROVIDER: S-EPMC9184808 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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

Mendelev Natalia N Zamojski Michel M Amper Mary Anne S MAS Cheng Wan Sze WS Pincas Hanna H Nair Venugopalan D VD Zaslavsky Elena E Sealfon Stuart C SC Ruf-Zamojski Frederique F

STAR protocols 20220607 2

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 chan ...[more]

PMID: 35693209

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Project description:BACKGROUND:The main bottleneck for genomic studies of tumors is the limited availability of fresh frozen (FF) samples collected from patients, coupled with comprehensive long-term clinical follow-up. This shortage could be alleviated by using existing large archives of routinely obtained and stored Formalin-Fixed Paraffin-Embedded (FFPE) tissues. However, since these samples are partially degraded, their RNA sequencing is technically challenging. RESULTS:In an effort to establish a reliable and practical procedure, we compared three protocols for RNA sequencing using pairs of FF and FFPE samples, both taken from the same breast tumor. In contrast to previous studies, we compared the expression profiles obtained from the two matched sample types, using the same protocol for both. Three protocols were tested on low initial amounts of RNA, as little as 100 ng, to represent the possibly limited availability of clinical samples. For two of the three protocols tested, poly(A) selection (mRNA-seq) and ribosomal-depletion, the total gene expression profiles of matched FF and FFPE pairs were highly correlated. For both protocols, differential gene expression between two FFPE samples was in agreement with their matched FF samples. Notably, although expression levels of FFPE samples by mRNA-seq were mainly represented by the 3'-end of the transcript, they yielded very similar results to those obtained by ribosomal-depletion protocol, which produces uniform coverage across the transcript. Further, focusing on clinically relevant genes, we showed that the high correlation between expression levels persists at higher resolutions. CONCLUSIONS:Using the poly(A) protocol for FFPE exhibited, unexpectedly, similar efficiency to the ribosomal-depletion protocol, with the latter requiring much higher (2-3 fold) sequencing depth to compensate for the relative low fraction of reads mapped to the transcriptome. The results indicate that standard poly(A)-based RNA sequencing of archived FFPE samples is a reliable and cost-effective alternative for measuring mRNA-seq on FF samples. Expression profiling of FFPE samples by mRNA-seq can facilitate much needed extensive retrospective clinical genomic studies.

Dataset Information

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

Publications

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

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