RT Journal Article
SR Electronic
T1 Lung, spleen and oesophagus tissue remains stable for scRNAseq in cold preservation
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 741405
DO 10.1101/741405
A1 Madissoon, E.
A1 Wilbrey-Clark, A.
A1 Miragaia, R.J.
A1 Saeb-Parsy, K.
A1 Mahbubani, K.
A1 Georgakopoulos, N.
A1 Harding, P.
A1 Polanski, K.
A1 Nowicki-Osuch, K.
A1 Fitzgerald, R.C.
A1 Loudon, K.W.
A1 Ferdinand, J.R.
A1 Clatworthy, M.R
A1 Tsingene, A.
A1 Van Dongen, S.
A1 Dabrowska, M.
A1 Patel, M.
A1 Stubbington, M.J.T.
A1 Teichmann, S.
A1 Stegle, O.
A1 Meyer, K.B.
YR 2019
UL http://biorxiv.org/content/early/2019/08/31/741405.abstract
AB Background The Human Cell Atlas is a large international collaborative effort to map all cell types of the human body. Single cell RNA sequencing can generate high quality data for the delivery of such an atlas. However, delays between fresh sample collection and processing may lead to poor data and difficulties in experimental design. Despite this, there has not yet been a systematic assessment of the effect of cold storage time on the quality of scRNAseqResults This study assessed the effect of cold storage on fresh healthy spleen, oesophagus and lung from ≥5 donors over 72 hours. We collected 240,000 high quality single cell transcriptomes with detailed cell type annotations and whole genome sequences of donors, enabling future eQTL studies. Our data provide a valuable resource for the study of these three organs and will allow cross-organ comparison of cell types.We see little effect of cold ischaemic time on cell viability, yield, total number of reads per cell and other quality control metrics in any of the tissues within the first 24 hours. However, we observed higher percentage of mitochondrial reads, indicative of cellular stress, and increased contamination by background “ambient RNA” reads in the 72h samples in spleen, which is cell type specific.Conclusions In conclusion, we present robust protocols for tissue preservation for up to 24 hours prior to scRNAseq analysis. This greatly facilitates the logistics of sample collection for Human Cell Atlas or clinical studies since it increases the time frames for sample processing.eQTLExpression quantitative trait lociscRNAseqSingle cell RNA sequencingHCAHuman Cell AtlasDMSODimethyl sulfoxideGTExGenotype-Tissue ExpressionCBTMCambridge Biorepository for Translational MedicineDCDDonation after cardiac deathDBDDonation after brainstem deathUWUniversity of WisconsinNRPNormothermic regional perfusionPCRPolymerase chain reactionRNARibonucleic acidDNADeoxyribonucleic acidPBSPhosphate buffered salineBSABovine serum albuminMACSMagnetic activated cell sortingFBSFoetal bovine serumHTAHuman Tissue AuthorityRECResearch Ethics CommitteePMIPost mortem intervalWGSWhole Genome SequencingSc-pseudo-bulkSingle-cell pseudo-bulk