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| Status |
Public on Aug 09, 2017 |
| Title |
Chip3-A3_S54 |
| Sample type |
SRA |
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| Source name |
CD4 T cell
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| Organism |
Homo sapiens |
| Characteristics |
tissue: cord blood
cell type: CD4 T cell
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| Treatment protocol |
Single-cell capture: Single-cells were captured using the C1™ Single-Cell Auto Prep System (Fluidigm, San Francisco, CA, USA). Two C1 arrays for mRNA sequencing (5-10µM) were used to isolate single cells. Briefly, 1000 cells were loaded onto each array after confirming cell suspension concentration using a disposable haemocytometer (C-Chip, InCyto, Chungcheongnam-do, Korea). The first array yielded 43 single cells, and the second 61 single cells. Excluded capture sites contained no cell, single cells with obvious cell debris or multiple cells.
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| Growth protocol |
CD4 T cells from cord blood samples (n=2) were isolated by cell sorting on a FACS Aria (BD) after staining with antibodies for CD4 negative selection (Miltenyi). Samples were then activated for 2.5hrs with PMA and ionomycin (P/I) before washing and collection.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
The SMARTer kit (Clontech, Mountain View, CA, USA) was used for cDNA generation. 1µL of the ERCC spike-in mix (Life Technologies, Carlsbad, CA, USA) was included in the cell lysis mix at a dilution of 1:1000. cDNA quantity was measured using the Qubit® 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA), and a subset of cDNAs were checked for quality using the Agilent 2200 Tapestation (Agilent Technologies, Waldbronn, Germany).
IL-8 qPCR: To determine expression levels of IL-8, real-time PCR was performed on all 104 single-cell cDNA samples using a sybr-green labelled primer/probe set Hs_CXCL8_1_SG QuantiTect Primer (Qiagen, Germantown, MD, USA). Two Taqman® endogenous control assays were used for normalisation purposes: GAPDH Hs99999905_m1 and EIF4A2 Hs00756996_g1 (Life Technologies). A standard curve of control cDNA from a sample known to express IL8 was run alongside the single-cell DNAs, and all samples were run in triplicate. Data was analysed using the ΔΔCt method. Library preparation and sequencing: A total of 96 single cells were taken forward for RNA-seq. Libraries were prepared using the Illumina Nextera XT Sample Preparation Kit (Illumina Inc., Cambridge, UK) with an input of 150pg of cDNA per sample. 75bp paired-end reads were generated for each library using the Illumina NextSeq®500.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Paired-end RNASeq reads from these samples were simultaneously aligned to the human genome (GRCh37/hg19) and ERCC92 spike-in control sequence using Tophat (v2.0.4) and Bowtie2 v2.0.0.beta6 with default settings.
Raw sequence counts mapped to genes or ERCC spike-in controls were obtained using htseq-count
Genome_build: hg19
Supplementary_files_format_and_content: consolidated_raw_counts_final - tab-delimited matrix of htseq-count results - gene name (rows) by sample names (columns)
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| Submission date |
Jul 21, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Deena L. Gibbons |
| E-mail(s) |
deena.gibbons@kcl.ac.uk
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| Phone |
+44 020 7188 0150
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| Organization name |
Kings College London
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| Department |
Dept. Immunology
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| Street address |
Great Maze Pond
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| City |
London |
| ZIP/Postal code |
SE1 9RT |
| Country |
United Kingdom |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE84686 |
Innate to adaptive: Human IFN-gamma producing CD4+ T cells can derive directly from CXCL8-producing recent thymic emigrants. |
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| Relations |
| BioSample |
SAMN05427354 |
| SRA |
SRX1968572 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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