PBMC fixation and processing for Chromium single-cell RNA sequencing

Journal of Translational Medicine
Jinguo ChenCHI Consortium

Abstract

Interest in single-cell transcriptomic analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. In almost all reported works investigators have used live cells, which introduces cell stress during preparation and hinders complex study designs. Recent studies have indicated that cells fixed by denaturing fixative can be used in single-cell sequencing, however they did not usually work with most types of primary cells including immune cells. The methanol-fixation and new processing method was introduced to preserve human peripheral blood mononuclear cells (PBMCs) for single-cell RNA sequencing (scRNA-Seq) analysis on 10× Chromium platform. When methanol fixation protocol was broken up into three steps: fixation, storage and rehydration, we found that PBMC RNA was degraded during rehydration with PBS, not at cell fixation and up to 3-month storage steps. Resuspension but not rehydration in 3× saline sodium citrate (SSC) buffer instead of PBS preserved PBMC RNA integrity and prevented RNA leakage. Diluted SSC buffer did not interfere with full-length cDNA synthesis. The methanol-fixed PBMCs resuspended in 3× SSC were successfully implemented into 10× Chromium standard scRNA-seq workflows wit...Continue Reading

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Methods Mentioned

BETA
single-cell sequencing
scRNA-Seq
Drop-seq
RNA-Seq
Assay
PCR
Chip
PCA
FACS

Software Mentioned

Cell Browser
Cell Ranger
Cell Ranger Pipeline
Partek Flow
bcl2fastq
STAR
Fluidigm
Seurat

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