|
| Status |
Public on May 07, 2013 |
| Title |
C29_WT |
| Sample type |
SRA |
| |
|
| Source name |
Bronchial Epithelial Cells
|
| Organism |
Homo sapiens |
| Characteristics |
donor: C29
genotype/variation: Untreated control
|
| Treatment protocol |
At confluence, Doxycycline 0.5 mg/ml was added for 24 h.
|
| Growth protocol |
Cells were seeded into plastic dishes coated with bovine collagen (PureCol, Advanced BioMatrix, 5005-B) in bronchial epithelial cell growth medium (BEGM) containing 0.11mM Ca++ and 25 ng/ml EGF. Confluent cultures were passaged to P2 in dishes without collagen.P2 cells at 40% confluence were infected with H2B-GFP or DN-GRHL2 lentivirus and 1 mg/ml puromycin added 48 h later.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Between 0.5-1 million cells were trypsinized, washed with PBS, pelleted and snap frozen. Total RNA was extracted using Qiagen RNeasy mini-columns with 1% β-mercaptoethanol in RLT buffer and a 15 min on column DNase digestion. mRNA was isolated using the Dynabeads mRNA Direct Kit (Invitrogen).
First strand cDNA was generated using SuperScript Vilo cDNA synthesis master mix (Invitrogen), and second strand cDNA was synthesized using E. coli DNA Polymerase I (New England Biolabs) with random hexamer primers. Double stranded cDNA was purified using 1.8X Agentcourt SPRI beads (Beckman Coulter). Double stranded cDNA was fragmented and sequencing adaptors added using Nextera transposase (Illumina) and indexed for sequencing by PCR as described previously. Samples were pooled in equimolar ratios and sequenced on an Illumina HiSeq 2000.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Base-calling was performed on instrument using CASAVA software
Reads were aligned to known RefSeq transcripts from hg19 using Bowtie version 0.12.8 and allowing up to 2 kb between paired reads.
Read counts were normalized and differential expression was called using Deseq (v. 1.10.1) by usiong a chi-squared test to compare fits to a model that contained donor and count (i.e. count ~ donor + condition) to a model that only contained donor information (i.e. count ~ donor).
P-values were converted to a False Discovery Rate using the method of Benjamini and Hochberg (1995)
Genome_build: hg19
Supplementary_files_format_and_content: Counts of the number of reads aligned to each RefSeq transcript.
|
| |
|
| Submission date |
Feb 27, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Timothy E Reddy |
| E-mail(s) |
tim.reddy@duke.edu
|
| Organization name |
Duke University
|
| Department |
Department of Biostatistics & Bioinformatics
|
| Lab |
ReddyLab
|
| Street address |
2347 CIEMAS, 101 Science Drive
|
| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27708 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE44718 |
Multiple roles for Grainyheadlike transcription factors in the establishment and maintenance of human mucociliary airway epithelium |
|
| Relations |
| SRA |
SRX246006 |
| BioSample |
SAMN01931868 |
