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Series GSE112845 Query DataSets for GSE112845
Status Public on Jul 25, 2018
Title PBMC Fixation and Processing for Chromium Single-Cell RNA Sequencing
Organism Homo sapiens
Experiment type Expression profiling by high throughput sequencing
Summary Background: Interest in single-cell whole transcriptome analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. In almost all reported works, investigators have used live cells which represent several inconveniences and limitations. Some recent cell fixation methods did not work with most primary cells including immune cells.
Methods: The methanol-fixation and new processing method was introduced to preserve PBMCs for single-cell RNA sequencing (scRNA-Seq) analysis on 10X Chromium platform.
Results: When methanol fixation protocol was broken up into three steps, we found that PBMC RNA was degraded during rehydration with PBS, not at cell fixation and up to three-month storage steps. Resuspension but not rehydration in 3X 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 3X SSC were successfully implemented into 10X Chromium standard scRNA-seq workflows with no elevated low quality cells and cell doublets. The fixation process did not alter the single-cell transcriptional profiles and gene expression levels. Major subpopulations classified by marker genes could be identified in fixed PBMCs at a similar proportion as in live PBMCs. This new fixation processing protocol was validated in CD8+ T cell and several other cell types.
Conclusions: We expect that the methanol-based cell fixation procedure presented here will substantially enable complex experimental design with primary cells at single cell resolution.
Overall design single cell RNA sequencing of PBMC, CD8+ T cell or KLM1 cell line after fixation with methonal and resuspension in SSC buffer
Contributor(s) Chen J, Cheung F, Tsang J
Citation(s) 30016977
Submission date Apr 09, 2018
Last update date Mar 26, 2019
Contact name Jinguo Chen
Phone 2406273273
Organization name NIH
Department NIAID
Lab Center for Human Immunology (CHI)
Street address 10 Center Drive
City Bethesda
State/province MD
ZIP/Postal code 20892
Country USA
Platforms (2)
GPL18573 Illumina NextSeq 500 (Homo sapiens)
GPL21290 Illumina HiSeq 3000 (Homo sapiens)
Samples (9)
GSM3087619 DTM-X_PBMC_live
GSM3087622 DTM-X_PBMC_methanol-3H+SSC
GSM3087624 DTM-X_PBMC_methanol-3W+SSC
BioProject PRJNA449292

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Supplementary file Size Download File type/resource
GSE112845_RAW.tar 202.4 Mb (http)(custom) TAR (of MTX, TSV)
Raw data are available in SRA
Processed data provided as supplementary file

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