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Sample GSM1943331 Query DataSets for GSM1943331
Status Public on Nov 20, 2015
Title Tgfb1_IL6_48hr_130715_T16_48h_SC46_144
Sample type SRA
 
Source name TGFB1_IL6-48h (single cell batch 6)
Organism Mus musculus
Characteristics strain: C57BL/6
cell type: CD4 T cells
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).
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.
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina Genome Analyzer II
 
Description Gaublomme_et_al_2015_single-cells_Tgfb1_IL6_48hr_130715_T16_48h_SC46_144
CD4+ T cells from C57BL/6 WT mice differentiated into Th17 cells in-vitro with Tgfb1 + IL6. Cells were profiled after 48hr
sample date (yymmdd): 130715
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)
 
Submission date Nov 17, 2015
Last update date May 15, 2019
Contact name Nir Yosef
E-mail(s) [email protected]
Phone (617) 714-7734
Organization name UC Berkeley
Department EECS
Lab Yosef
Street address Berkeley
City Berkeley
State/province CA
ZIP/Postal code 94720
Country USA
 
Platform ID GPL9250
Series (2)
GSE74833 Identification of novel regulators of Th17 cell pathogenicity by single-cell genomics
GSE75110 Single-cell transcriptional profiling of Th17 cells, differentiated in vitro for 48h [TGFB1_IL6-48h]
Relations
BioSample SAMN04273466
SRA SRX1435732

Supplementary file Size Download File type/resource
GSM1943331_single-cells_Tgfb1_IL6_48hr_130715_T16_48h_SC46_144.genes.fpkm_tracking.gz 907.2 Kb (ftp)(http) FPKM_TRACKING
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data are available in SRA

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