|
| Status |
Public on Mar 01, 2022 |
| Title |
WTBE ALI rep1 (ATAC-seq) |
| Sample type |
SRA |
| |
|
| Source name |
Epithelial cells
|
| Organism |
Homo sapiens |
| Characteristics |
culture condition: Air-liquid interface
cell line: WTBE (CFBE41o- cells stably expressing wild-type CFTR)
cell type: Cystic fibrosis (CF) bronchial epithelial cells
|
| Treatment protocol |
For transwell-cultured (ALI) condition, cells were seeded on Millicell cell culture insert (Millipore) placed in 24-well plates. Media on the apical surface was removed daily until complete confluency (dry membrane) and basolateral media were replaced every other two days. Confluent cells were cultured for at least another 10days for polarization. For submerged cells, cells were also cultured in 24well plates and cultured till fully confluent.
|
| Growth protocol |
Human bronchial epithelial CFBE41o- cells stably expressing F508del-CFTR (CFBE) or wild-type CFTR (WTBE) were cultured in MEM medium, supplemented with 10% fetal bovine serum (Hyclone), 0.5% Penicillin/Streptomycin and 2mM L-glutamine. 0.5µg/mL puromycin (InvivoGen) were added for WTBE while 25µg/mL puromycin for CFBE cells to select stably transfected cells. Cells were maintained in a humidified chamber with 5% CO2 at 37C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
60,000 cells (>95% viability) were trypsinized and lysed in 50L cold lysis buffer (10mm Tris-HCl, 10mM NaCl, 3mM MgCl2, 0.1% NP40, 10% Tween-20, and 0.01% Digitonin) and incubated on ice for 5min. Then 1mL wash buffer (10mm Tris-HCl, 10mM NaCl, 3mM MgCl2 and 10% Tween-20) was added and tube were inverted 3 times gently.
Nuclei were pelleted at 500g for 5min at 4°C and resuspended in 50 µL of transposition mixture (TDE1 tagment DNA enzyme and TD buffer, illumina) and incubated for 30 min at 37 °C. Isolated DNA samples were purified using DNA Clean and Concentrator-5 (Zymo Research), which can be stored in -20°C. Purified DNA products were pre-amplified for 5 cycles with NEBNext High-Fidelity 2x PCR MasterMix (NEB). Additional cycles were added based on qPCR results of partially-amplified library. PCR reactions were purified and qualified using an Agilent High Sensitivity DNA Bioanalyzer Kit (Agilent). The libraries were quantified using a sensitive fluorescence dye-based Qubit dsDNA HS Assay Kit (ThermoFisher). Libraries were pooled at a final concentration of 650pM
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
| |
|
| Description |
ALI_WTBE1
|
| Data processing |
Paired-end reads were aligned to the human hg19 database (GRCh37.p13) using BWA.
The mitochondrial reads and reads with mapQuality <30 were removed. Duplicate reads were cleaned by MarkDuplicates tool. The narrowpeaks were called using MACS2 from the converted BED files. Normalized bigwig files were generated by Wig/BedGraph-to-BigWig tool on Galaxy platform
Peak annotations and feature distributions were performed by ChipSeeker (Bioconductor) and KEGG pathways were analyzed by PathfindR in R studio.
Genome_build: hg19
Supplementary_files_format_and_content: callpeak normalized reads
|
| |
|
| Submission date |
Nov 01, 2021 |
| Last update date |
Mar 01, 2022 |
| Contact name |
Jay Kolls |
| Organization name |
Tulane University
|
| Department |
Medicine
|
| Lab |
Center for Translational Research in Infection and Inflammation
|
| Street address |
1430 Tulane Ave
|
| City |
New Orleans |
| State/province |
LA |
| ZIP/Postal code |
70112 |
| Country |
USA |
| |
|
| Platform ID |
GPL30173 |
| Series (2) |
| GSE186918 |
ATACseq of CFBE41o- stably expressing wild-type CFTR (WTBE) and F508del-CFTR (CFBE) cells |
| GSE186920 |
Multi-omics comparisons between CFBE41o- cells stably expressing wide-type CFTR and F508del-mutant CFTR |
|
| Relations |
| BioSample |
SAMN22818350 |
| SRA |
SRX12877111 |
