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Series GSE130882 Query DataSets for GSE130882
Status Public on May 24, 2019
Title Evaluation of ultra-low input RNA sequencing for the study of human T cell transcriptome
Organism Homo sapiens
Experiment type Expression profiling by high throughput sequencing
Summary Deeper understanding of T cell biology is crucial for the development of new therapeutics. Human naïve T cells have low RNA content and their numbers can be limiting; therefore we set out to determine the parameters for robust ultra-low input RNA sequencing. We performed transcriptome profiling at different cell inputs and compared three protocols: Switching Mechanism at 5’ End of RNA Template technology (SMART) with two different library preparation methods (Nextera (SMART_Nxt) and Clontech (SMART_CC)), and AmpliSeq technology. As the cell input decreased the number of detected coding genes decreased with SMART, while stayed constant with AmpliSeq. However, SMART enables detection of non-coding genes, which is not feasible for AmpliSeq. The detection is dependent on gene abundance, but not transcript length. The consistency between technical replicates and cell inputs was comparable across methods above 1K but highly variable at 100 cell input. Sensitivity of detection for differentially expressed genes decreased dramatically with decreased cell inputs in all protocols, support that additional approaches, such as pathway enrichment, are important for data interpretation at ultra-low input. Finally, T cell activation signature was detected at 1K cell input and above in all protocols, with AmpliSeq showing better detection at 100 cells.
Overall design Primary human naïve CD4 T cells were treated with a-CD3 only or a-CD3 and B7-1 Fc, which stimulate T cell activation, for 2 hours. Next, the cells were collected and serially diluted to achieve 100K, 5K, 1K and 100 cells. Finally, RNA was extracted and transcriptome library was generated with two different protocols: SMART-Seq and AmpliSeq. For SMART-Seq technology, two different library preparation kits were utilized: Nextera Library Preparation Kit (SMART_Nxt) and Clontech Library Preparation kit (SMART_CC)
Contributor(s) Wang J, Raja R, Rieder S
Citation(s) 31186477
Submission date May 08, 2019
Last update date Jun 24, 2019
Contact name Jingya Wang
Phone 3013980977
Organization name Astrazeneca
Department Early respiratory and immunology
Street address 1 Medimmune Way
City Gaithersburg
State/province Maryland
ZIP/Postal code 20878
Country USA
Platforms (3)
GPL11154 Illumina HiSeq 2000 (Homo sapiens)
GPL17303 Ion Torrent Proton (Homo sapiens)
GPL18573 Illumina NextSeq 500 (Homo sapiens)
Samples (48)
GSM3755579 CD3_100k_rep1_SMART_Nxt
GSM3755580 CD3_100k_rep2_SMART_Nxt
GSM3755581 CD3_5k_rep1_SMART_Nxt
BioProject PRJNA541904
SRA SRP197074

Download family Format
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Supplementary file Size Download File type/resource
GSE130882_RAW.tar 4.5 Mb (http)(custom) TAR (of TXT)
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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