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| Status |
Public on Mar 04, 2021 |
| Title |
NS8 NICD3 day 3 |
| Sample type |
SRA |
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| Source name |
primary human bronchial epithelial cells (HBECs)
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| Organism |
Homo sapiens |
| Characteristics |
patient: nonsmoker donor 2
treatment: Lenti-NICD3
time point: day 3
cell type: HBEC (Primary human bronchial epithelial cells )
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| Treatment protocol |
HBECs were infected with either Lenti-Control or Lenti-NICD3 at a multiplicity of infection of 50 (5 x 106 viral genomes/1 x 105 cells) at the time of seeding the cells on ALI to ensure that >90% of cells were infected (GFP+) with each virus. To aid virus infection, the BLEAM media was supplemented with 2 μg/ml of polybrene (catalog number TR-1003-G, Sigma Aldrich). The next day, fresh media was added to the wells and the standard ALI protocol was continued until the day of harvest.
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| Growth protocol |
Primary human bronchial epithelial cells (HBECs) from nonsmokers cultured on air-liquid interface (ALI) for up to 7 days. Briefly, 1 x 105 cells in 100 µl of BronchiaLifeTM epithelial airway medium (BLEAM) (Cat# LL-0023, Lifeline® Cell Technology, Oceanside, CA, USA) were seeded in the apical chamber of a 0.4 µm pore-sized Transwell® insert (Cat# 3470, Corning®, Corning, NY, USA) pre-coated with human type IV collagen (Cat# C7521, Sigma Aldrich, St. Louis, MO, USA) with 1 ml of BLEAM in the basolateral chamber (ALI day -2). The following day, fresh BLEAM media was replaced in the apical and basolateral chambers (100 µl and 1 ml, respectively). Following two days of submerged culture, media from the apical chamber was removed to expose the cells to air (ALI day 0), and 1 ml of HBTEC ALI differentiation medium (Cat# LM-0050, Lifeline® Cell Technology) supplemented with Penicillin (100 I.U./ml) - Streptomycin (100 µg/ml) was added to the basolateral chamber. The media was replaced every other day and the cells allowed to differentiate for up to 7 days.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted via direct lysis of cells in the ALI well using the PureLinkTM RNA mini kit (Cat# 12183018A, Thermo Scientific, Waltham, MA, USA) and included DNase treatment (Cat# 12185-010, Thermo Scientific) on the column to remove contaminating genomic DNA. RNA concentrations were measured with a NanoPhotometer® N60 (Implen, Westlake Village, CA, USA)
RNA-sequencing was performed on a NextSeq 500 Flowcell, High SR75 (Illumina, San Diego, CA, USA) following library preparation using the QuantSeq 3′ mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen, Vienna, Austria). The library preparation and sequencing were performed by the Clinical Genomics Core at the Oklahoma Medical Research Foundation
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Raw sequencing reads (in a FASTQ format) were trimmed of residual adaptor sequences using Scythe software. Low quality bases at the beginning or the end of sequencing reads were removed using sickle then the quality of remaining reads was confirmed with FastQC.
Reads were aligned to the Homo sapiens genome reference (GRCh38/hg38) using the TopHat component of Tuxedo Suite software, then cuffquant and cuffdiff were used for gene-level read counting and differentially expression analysis.
Genome_build: GRCh38/hg38
Supplementary_files_format_and_content: tab-delimited text file includes locus, REFSEQ isoform id, Gene name and raw read counts for each transcript, in each sample
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| Submission date |
Mar 03, 2021 |
| Last update date |
Mar 04, 2021 |
| Contact name |
Jonathan Wren |
| E-mail(s) |
Jonathan-Wren@omrf.org
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| Phone |
(405) 271-6989
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| Organization name |
OMRF
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| Department |
Genes & Human Disease Research Program
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| Street address |
825 N.E. 13th Street
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| City |
Oklahoma City |
| State/province |
OK |
| ZIP/Postal code |
73104 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE168126 |
The NOTCH3 Downstream Target HEYL Is Required for Efficient Human Airway Basal Cell Differentiation [RNA-Seq] |
| GSE168128 |
The NOTCH3 Downstream Target HEYL Is Required for Efficient Human Airway Basal Cell Differentiation |
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| Relations |
| BioSample |
SAMN18121139 |
| SRA |
SRX10218252 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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