High-throughput Preparation of DNA, RNA, and Protein from Cryopreserved Human iPSCs for Multi-omics Analysis

Curr Protoc Stem Cell Biol. 2020 Sep;54(1):e114. doi: 10.1002/cpsc.114.

Abstract

We describe the procedure to isolate genomic DNA, RNA, and protein directly from cryopreserved induced pluripotent stem cell (iPSC) vials using commercially available solid-phase extraction kits, and we report the relationship between macromolecule yields and experimental and storage factors. Sufficient quantities of DNA, RNA, and protein are recoverable from as low as 1 million cryopreserved cells across 728 distinct iPSC lines suitable for whole-genome sequencing, RNA sequencing, and mass spectrometry experiments. Nucleic acids extracted from iPSC stocks cryopreserved up to 4 years maintain sufficient quantity and integrity for downstream analysis with minimal genomic DNA fragmentation. An expected positive correlation exists between cell count and DNA or RNA yield, with comparable yields recovered between cells across different cryostorage timespans. This article provides an effective way to simultaneously isolate iPSC biomolecules for multi-omics investigations. © 2020 Wiley Periodicals LLC. Basic Protocol 1: QIAshredder and AllPrep DNA/RNA/protein mini kit extraction and subsequent DNA quantification and quality analysis Basic Protocol 2: Broad-range RNA quantification and quality assay using QuBit 4 Fluorometer and associated kits.

Keywords: biobank; cryopreservation; induced pluripotent stem cells; multimodal omics analysis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cryopreservation*
  • DNA / isolation & purification*
  • Fluorometry
  • Genomics*
  • High-Throughput Screening Assays / methods*
  • Humans
  • Induced Pluripotent Stem Cells / metabolism*
  • Linear Models
  • Proteins / isolation & purification*
  • RNA / isolation & purification*

Substances

  • Proteins
  • RNA
  • DNA