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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Nov 20, 2015 |
| Title |
Tgfb1_IL6-IL17A_POS_48hr_140519_T16_IL17A_48h_SC29_408 |
| Sample type |
SRA |
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| Source name |
TGFB1_IL6-48h-IL-17A/GFP+ (single cell batch 8)
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: CD4 T cells
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| Treatment protocol |
CD4+ T cells were purified from spleen and LNs using anti-CD4 microbeads (Miltenyi Biotech) and then stained in PBS with 1% FCS for 20 min at room temperature with anti-CD4-PerCP, anti-CD62L-APC and anti-CD44-PE antibodies (all Biolegend). Naive CD4+CD62lhighCD44low T cells were sorted using a BD FACSAria cell sorter. Sorted cells were activated with plate-bound anti-CD3 (2 μg/ml) and anti-CD28 (2 μg/ml) in the presence of cytokines. For Th17 differentiation, the following reagents were used: 2 ng/ml recombinant human TGF-β1 (Miltenyi Biotec), 25 ng/ml recombinant mouse IL-6 (Miltenyi Biotec), 20 ng/ml recombinant mouse IL-23 (R&D Biosystems) and 20 ng/ml recombinant mouse IL-1β (Miltenyi Biotec).
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| Growth protocol |
C57BL/6 WT and CD4-/-(2663) mice were obtained from Jackson Laboratory. IL-17A–GFP+ mice were obtained from Biocytogen. All animals, unless noted otherwise, were housed and maintained in a conventional pathogen-free facility at the Harvard Institute of Medicine in Boston. All experiments were performed in accordance to the guidelines outlined by the Harvard Medical Area Standing Committee on Animals at the Harvard Medical School.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were washed, stained and sorted for CD3 (Biolegend), CD4 (Biolegend), 7AAD and IL-17A-GFP+.
Cell lysis and SMART-Seq (Ramskold et al., 2012) whole transcriptome amplification (WTA) was performed on the C1 chip using the Fluidigm C1 Single-Cell Auto Prep System (C1 System) with the SMARTer Ultra Low RNA Kit for Illumina Sequencing (Clontech). WTA products were harvested from the C1 chip, and cDNA libraries were prepared using Nextera XT DNA Sample preparation reagents (Illumina) as per manufacturer’s recommendations, with minor modifications. Specifically, reactions were run at ¼ the recommended volume, the tagmentation step was extended to 10 minutes, and the extension time during the PCR step was increased from 30s to 60s. After the PCR step, all 96 samples were pooled without library normalization, cleaned twice with 0.9x AMPure XP SPRI beads (Beckman Coulter), and eluted in buffer TE. The pooled libraries were quantified using Quant-IT DNA High-Sensitivity Assay Kit (Invitrogen) and examined using a high sensitivity DNA chip (Agilent). Finally, samples were sequenced deeply using either a HiSeq 2000 or a HiSeq 2500 sequencer.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
Gaublomme_et_al_2015_single-cells_Tgfb1_IL6-IL17A_POS_48hr_140519_T16_IL17A_48h_SC29_408
CD4+ T cells from IL-17A–GFP+mice differentiated into Th17 cells in-vitro with Tgfb1 + IL6. GFP+ cells were sorted and profiled after 48hr
sample date (yymmdd): 140519
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| Data processing |
base calling software
RNA-seq reads were aligned to the NCBI Build 37 (UCSC mm9) of the mouse genome using TopHat (Trapnell et al., 2009). The resulting alignments were processed by Cufflinks to evaluate the expression of transcripts from RefSeq.
Genome_build: mm9
Supplementary_files_format_and_content: Tab delimited text. Standard cufflinks output with columns for: (tracking_id, class_code, nearest_ref_id, gene_id, gene_short_name, tss_id, locus, length, coverage, FPKM, FPKM_conf_lo, FPKM_conf_hi, FPKM_status)
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| Submission date |
Nov 17, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Nir Yosef |
| E-mail(s) |
niryosef@berkeley.edu
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| Phone |
(617) 714-7734
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| Organization name |
UC Berkeley
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| Department |
EECS
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| Lab |
Yosef
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| Street address |
Berkeley
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| City |
Berkeley |
| State/province |
CA |
| ZIP/Postal code |
94720 |
| Country |
USA |
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| Platform ID |
GPL9250 |
| Series (2) |
| GSE74833 |
Identification of novel regulators of Th17 cell pathogenicity by single-cell genomics |
| GSE75111 |
Single-cell transcriptional profiling of Th17 cells, differentiated in vitro for 48h [TGFB1_IL6-48h-IL-17A/GFP+] |
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| Relations |
| BioSample |
SAMN04273659 |
| SRA |
SRX1435880 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1943476_single-cells_Tgfb1_IL6-IL17A_POS_48hr_140519_T16_IL17A_48h_SC29_408.genes.fpkm_tracking.gz |
904.4 Kb |
(ftp)(http) |
FPKM_TRACKING |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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