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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 07, 2013 |
| Title |
Non-targeting control 3_2_12__48h_NT__2_4 |
| Sample type |
SRA |
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| Source name |
Th17 cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: Th17 cells
batch: 3_2_12
treatment: Non-targeting control
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| Treatment protocol |
Cd4+ T cells were purified from spleen and lymph nodes using anti-CD4 microbeads (Miltenyi Biotech) 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, CA). Naïve Cd4+ Cd62lhigh Cd44low T cells were sorted using the BD FACSAria cell sorter. 4 x 4 mm silicon nanowire (NW) substrates were prepared and coated with 3 μL of a 50 μM pool of four siGENOME siRNAs (Dharmcon) in 96 well tissue culture plates. Briefly, 150,000 sorted naïve T cells were seeded on siRNA-laced NWs in 10 μL of complete media and placed in a cell culture incubator (370C, 5% CO2) to settle for 45 minutes before full media addition. These samples were left undisturbed for 24 hours to allow target transcript knockdown. Afterward, siRNA-transfected T cells were activated with αCd3/Cd28 dynabeads (Invitrogen), according to the manufacturer’s recommendations, under Th17 polarization conditions (2ng/mL rhTGF-β1 (Miltenyi Biotec), 25ng/mL rmIl-6 (Miltenyi Biotec)).
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| Growth protocol |
C57BL/6 mice were purchased from The Jackson Laboratory. All animals were housed and maintained in a conventional pathogen-free facility at the Harvard Institute of Medicine in Boston, MA. All experiments were performed in accordance to the guidelines outlined by the Harvard Medical Area Standing Committee on Animals at the Harvard Medical School (Boston, MA).
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| Extracted molecule |
total RNA |
| Extraction protocol |
48h post-activation, culture media was removed from each well and samples were gently washed with 100 μL of PBS before being lysed in 20 μL of buffer TCL (Qiagen) supplemented with 2-mercaptoethanol (1:100 by volume). After mRNA was harvested in Turbocapture plates (Qiagen) and converted to cDNA using Sensiscript RT enzyme (Qiagen), qRT-PCR was used to validate both knockdown levels and phenotypic changes relative to 8-12 non-targeting siRNA control samples. A 60% reduction in target mRNA was used as the knockdown threshold.
Validated single stranded cDNAs from the NW-mediated knockdowns were converted to double stranded DNA using the NEBNext mRNA Second Strand Synthesis Module (New England BioLabs) according to the manufacturer’s recommendations. The samples were then cleaned using 0.9x SPRI beads (Beckman Coulter). Libraries were prepared using the Nextera XT DNA Sample Prep Kit (Illumina), quantified, pooled, and then sequenced on the HiSeq 2500 (Illumnia) to an average depth 20M reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer |
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| Description |
Non-targeting control
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| Data processing |
RNA-seq reads were aligned to the NCBI Build 37 (UCSC mm9) of the mouse genome using Bowtie
We ran RSEM v1.1138 with default parameters on these alignments to estimate expression levels
Genome_build: mm9
Supplementary_files_format_and_content: text files produced by RSEM; data is in transcirpts per million (TPM)
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| Submission date |
Jan 31, 2013 |
| 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 |
GPL9185 |
| Series (2) |
| GSE43948 |
Reconstruction of the dynamic regulatory network that controls Th17 cell differentiation by systematic perturbation in primary cells (RNA-Seq) |
| GSE43970 |
Reconstruction of the dynamic regulatory network that controls Th17 cell differentiation by systematic perturbation in primary cells |
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| Relations |
| SRA |
SRX220918 |
| BioSample |
SAMN01908554 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1074858_3_2_12_48h_NT_2_4.rsem.txt.gz |
220.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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