|
| Status |
Public on Nov 11, 2024 |
| Title |
H14 V1 N |
| Sample type |
SRA |
| |
|
| Source name |
primary bronchial epithelial cells
|
| Organism |
Homo sapiens |
| Characteristics |
donor: Healthy controls
collection method: direct bronchoscopy
type: in vivo
tissue: Healthy bronchial epithelium
|
| Treatment protocol |
The brushes which consisted predominantly (>95%) of PBECs, were pooled (2 brushes for each sample) and centrifuged at 1240 rpm (Rotanta 460S) for 10 minutes at 4°C.
|
| Growth protocol |
The bronchial brushes were obtained from bronchoscopy
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The pellet was dissolved in 1 ml of TRIzol™ and stored at -80°C until RNA was isolated. After all samples were obtained, they were thawed at room temperature and, after 200 µl of chloroform was added, shaken vigorously for 30 seconds. The samples were kept at RT for 10 mins and centrifuged at 16,000g for 15 mins at 4°C. The aqueous phase was cleaned up and concentrated with protocol 5.3 using the Nucleospin® RNA XS extraction kit (Macherey-Nagel). No carrier RNA was added as it would interfere with RNA sequencing.
The NEBNext Ultra Directional RNA library prep kit for Illumina was used to process the samples. The samples preparation was performed according to the protocol “NEBNext Ultra Directional RNA Library Prep Kit for Illumina” (NEB #7420S/L). Briefly, oligo-dT magnetic beads were used to isolate mRNA from total RNA. cDNA synthesis was performed after fragmentation of this mRNA. This was used for ligation with sequencing adapters followed by PCR amplification of the resulting product. The quality and yield after sample preparation was measured with the fragment analyser. The size of the resulting products was consistent with the expected size distribution between 300-500 base pairs. To evaluate the quality of the library preparation and kits used, the raw data was sampled and mapped to annotated genomic references. Mapping positions were classified as intragenic, exonic, intergenic, intronic and rRNA. Clustering and DNA sequencing was performed according to the manufacture’s protocol using the Illumina NextSeq 500. A concentration of 1.6pM was used as input. The reads were trimmed for adapter sequences using Trimmomatic v0.30 before the alignment. Presumed adapter sequences were removed from the read when the bases matched a sequence in the adapter sequence set (TruSeq adapters) with 2 or less mismatches and an alignment score of at least 12.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
102939-001-019
|
| Data processing |
The reference of ‘Homo_sapiens.GRCh37.75’ was used to align the reads. The mapping to the reference sequence was done using a short reader aligner based on Burrows-Wheeler transform.
Clustering and RNA sequencing was performed according to the manufacture’s protocol using the Illumina NextSeq 500
The reads were trimmed for adapter sequences using Trimmomatic v0.30 before the alignment
The read counts were loaded into the DESeq2 package, a statistical package within the R/BioConductor platform to determine the differentially expressed genes (paired)
Image analysis, base calling and quality check was performed with Illumina data analysis pipeline RTA v2.4.11 and Bcl2fastq v2.17.
Genome_build: hg19
Supplementary_files_format_and_content: count files: curated counts of genes in each sample
|
| |
|
| Submission date |
Nov 11, 2020 |
| Last update date |
Nov 11, 2024 |
| Contact name |
Abilash Ravi |
| E-mail(s) |
a.ravi@lumc.nl
|
| Phone |
+31 616451246
|
| Organization name |
Leiden University Medical Center
|
| Department |
Department of Pulmonology
|
| Street address |
Albinusdreef 2
|
| City |
Leiden |
| ZIP/Postal code |
2333 ZA |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE161245 |
Comparison of bronchial epithelial cells obtained by bronchoscopy from mild asthma patients, moderate asthma patients, severe asthma patients and healthy controls |
|
| Relations |
| BioSample |
SAMN16770325 |
| SRA |
SRX9474161 |
