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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Nov 20, 2015 |
| Title |
PLZP-KO-No_cytokines_added-48h (population, rep1) |
| Sample type |
SRA |
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| Source name |
CD4 T cells
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| Organism |
Mus musculus |
| Characteristics |
strain: B6x129S1
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, or provided by collaborator as sorted cells, shipped on ice overnight. 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. In addition, spleens and lymph nodes from GPR65−/− mice were provided by Li Yang (IACUC protocol: J195b), originally generated by Dr. Owen Witte. PLZP−/− mice were provided by Pier Paolo Pandolfi at the Beth Israel Deaconess Medical Center (IACUC protocol: 082-2014), and TOSO−/− mice were generously provided by John Coligan at the National Institute of Allergy and Infectious Diseases.
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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+.
population controls were generated by extracting total RNA using a RNeasy plus Micro RNA kit (Qiagen) according to manufacturer’s recommendations. Subsequently, 1 μL of RNA in water was added to 2 μL of lysis reaction mix, thermocycled using cycling conditions I described in Supplementary Experimental Procedures in corresponding publication. Next, 4 μL of the RT Reaction Mix were added, and the mixture was thermocycled using cycling conditions II. Finally, 1 μL of the total RT reaction was added to 9 μL of PCR mix, and that mixture was thermocycled using cycling conditions III. Products were quantified, diluted to 0.125 ng/ μL, and libraries were prepared, cleaned, and tested as described in Supplementary Experimental Procedures in corresponding publication.
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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_populations_invitro_knockouts_PLZP-KO-Th0-48-1
sample date (yymmdd): 140224
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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 |
| GSE75104 |
Population transcriptional profiling of KO or WT cells,, differentiated in vitro for 48-96h towards Th17 cells |
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| Relations |
| BioSample |
SAMN04273384 |
| SRA |
SRX1435162 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1942780_populations_invitro_knockouts_PLZP-KO-Th0-48-1.genes.fpkm_tracking.gz |
905.7 Kb |
(ftp)(http) |
FPKM_TRACKING |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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