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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Nov 20, 2015 |
| Title |
EAE-IL17A-LN_140107_eae_il17a_ln_SC7_255 |
| Sample type |
SRA |
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| Source name |
EAE-LN-IL-17A/GFP+ (single cell batch 3)
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/16
cell type: CD4 T cells
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| Treatment protocol |
For active induction of EAE, mice were immunized by subcutaneous injection of 100 μg MOG35-55 (MEVGWYRSPFSRVVHLYRNGK) in CFA, and then received 200 ng pertussis toxin intraperitoneally (List Biological Laboratory) on days 0 and 2. Mice were monitored and were assigned scores daily for development of classical and atypical signs of EAE according to the following criteria (Jager et al., 2009): 0, no disease; 1, decreased tail tone or mild balance defects; 2, hind limb weakness, partial paralysis or severe balance defects that cause spontaneous falling over; 3, complete hind limb paralysis or very severe balance defects that prevent walking; 4, front and hind limb paralysis or inability to move body weight into a different position; 5, moribund state.
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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 |
At the peak of disease, T cells were collected from the draining LNs and the CNS. For isolation from the CNS, mice were perfused through the left ventricle of the heart with cold PBS. The brain and the spinal cord were flushed out with PBS by hydrostatic pressure. CNS tissue was minced with a sharp razor blade and digested for 20 min at 37°C with collagenase D (2.5 mg/ml; Roche Diagnostics) and DNaseI (1 mg/ml; Sigma). Mononuclear cells were isolated by passage of the tissue through a cell strainer (70 mm), followed by centrifugation through a Percoll gradient (37% and 70%). After removal of mononuclear cells, the lymphocytes 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_EAE-IL17A-LN_140107_eae_il17a_ln_SC7_255
IL17-GFP+ T cells collected from the draining LN of EAE-induced mice. Cells were colected at the peak of disease (14 days post immunization).
sample date (yymmdd): 140107
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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 Scripture (Guttman et al., 2010) to evaluate the expression of transcripts from RefSeq (Pruitt et al., 2007).
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files output by the scripture software (Guttman et al. 2010); columns: (1--3) genomic location (4) gene name (5) RPKM
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| Submission date |
Nov 17, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Nir Yosef |
| E-mail(s) |
[email protected]
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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 |
| GSE75108 |
Single-cell transcriptional profiling of Th17 cells, harvested at peak of disease in EAE from LN [EAE-LN-IL-17A/GFP+] |
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| Relations |
| BioSample |
SAMN04273929 |
| SRA |
SRX1435480 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1943088_single-cells_EAE-IL17A-LN_140107_eae_il17a_ln_SC7_255.genes.fpkm_tracking.gz |
917.8 Kb |
(ftp)(http) |
FPKM_TRACKING |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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