 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jun 08, 2016 |
| Title |
Th2 d13 unstimulated mRNA rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Th2
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Th2
passages: 13 days
|
| Treatment protocol |
Naïve human CD4+ T-cells (CD4+CD45RA+CD45RO-CD25-CCD7+) were isolated from healthy donors by negative immunomagnetic selection (Miltenyi Biotec) and activated for 72 hours by plate bound anti-CD3 and anti-CD28 antibodies (2 µg/ml, R&D) and then cultured for 10 days with rhIL-2 (Biolegend, 10 ng/ml). Conditions for T ell polarisation were: rhIL-12 (10 ng/ml, Biolegend) and anti-IL-4 (10 μg/ml, R&D) for Th1, rhIL-4 (10 ng/ml, Biolegend) and anti-IFN-γ (10 μg/ml, R&D) for Th2. For ChIP, cells were formaldehyde crosslinked on day 13 either before (unstimulated) or after (restimulated) treatment with anti-CD3 and anti-CD28 antibodies (2 μg/ml). For H3K4me3 ChIP-Seq, CD4+ naive T-cells were differentiated into either Th1 or Th2 cells in vitro for 28 days, following an established protocol. Cells were activated for 4 hrs with 5 ng/ml PMA (Sigma) and 500 ng/ml ionomycin (CN Biosciences) and assessed for intracellular cytokine staining.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA naïve CD4+ cells and day 13 resting and restimulated Th1 and Th2 cells was obtained from samples previously used for microarray analysis.
Poly-adenylated RNA was purified from 1 mg of total RNA using the Qiagen Oliogtex kit and ribosomal RNA was depleted from total RNA using Ribo-Zero Gold (EpiCentre), as per manufacturers instructions. 5’ cap structures were removed with tobacco acid pyrophosphatase (TAP, Life Technologies) and RNA fragmented with potassium acetate (100mM) and magnesium acetate (30mM) at 94oC for 3 mins. RNA was then repaired with Antartic phosphatase and PNK, according to the instructions in the Illumina Directional mRNA-Seq Sample Prep Guide. Libraries were generated using the NEBNext Multiplex Small RNA Library Prep Set for Illumina kit, with 14 cycles of PCR amplification, and products between 130bp and 350bp gel purified. Libraries were quantified by qPCR (Library Quantification Kit, Kapa Biosystems) and average fragment size determined on an Agilent 2100 Bioanalyzer using DNA HS assays to pool libraries at equimolar concentrations.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
mRNA
|
| Data processing |
Basecalls performed using CASAVA
paired-end fastq files quality filtered using fastq-mcf -S -t 0.0001 -l 20
qc paired-end fastq files mapped using tophat v2.0.9 using tophat2 --b2-very-sensitive -g 2 -p 4 --library-type fr-secondstrand
mapped data run in cufflinks v2.1.1 with default settings (masking for rRNA, snRNA, Mt_rRNA, snoRNA and miRNA listed in Gencode v19 gene annotation)
Genome_build: hs19
Supplementary_files_format_and_content: Transfrags predicted by Cufflinks, out put in GTF format
|
| |
|
| Submission date |
Oct 18, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Richard Jenner |
| E-mail(s) |
r.jenner@ucl.ac.uk
|
| Organization name |
UCL Cancer Institute
|
| Department |
Cancer Biology
|
| Lab |
Regulatory Genomics
|
| Street address |
72 Huntley Street
|
| City |
London |
| ZIP/Postal code |
WC1E 6BT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE62483 |
T-bet recruits P-TEFb to super-enhancers to regulate T helper cell differentiation (RNA-Seq) |
| GSE62486 |
T-bet recruits P-TEFb to super-enhancers to regulate T helper cell differentiation |
|
| Relations |
| BioSample |
SAMN03120381 |
| SRA |
SRX735338 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1527732_140325_CE19_Hs_Hs_IJ1_Th2_d13_US_mRNA_CAGATC_L005_R1_001_QC_tophat2_out_auto_accepted_hits_cufflinks_out_transcripts.gtf.gz |
6.1 Mb |
(ftp)(http) |
GTF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
