|
| Status |
Public on Sep 03, 2020 |
| Title |
Wild Type (PacBio) |
| Sample type |
SRA |
| |
|
| Source name |
KBM-7 derived near-haploid cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HAP1
genotype: wild type
|
| Growth protocol |
The cell lines were cultured in IMDM medium (Quality Biological) supplemented with 10% (v/v) Tet System Approved fetal calf serum (Takara Bio/previously Clontech), 100 U/mL penicillin, 100 µg/mL streptomycin (Pen/Strep) (Thermo Fisher Scientific), at 37°C in an incubator containing 5% CO2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was cleaned via Agencourt RNA Clean XP (Beckman Coulter) beads and resuspended in Buffer EB (Qiagen). Quality and quantity was assessed via BioPhotometer (Eppendorf) and Agilent RNA 6000 Pico kit on the 2100 Bioanalyzer (Agilent Technologies).
SMRTbell libraries were generated using Pacific Bioscience’s no-size selection Iso-Seq Template Preparation guide following the manufacture’s recommendations. Input amounts for generating cDNA (Takara Bio) was 1000ng and PCR was optimized at 10 cycles for subsequent large-scale PCR. Fraction 1 and 2 of the cleaned-up products were quantified via Qubit (Thermo Fisher Scientific) fluorometric assay and equal molar quantities were combined resulting in over 1 µg cDNA going forward. Final SMRTbell libraries were quantified using Qubit and qualitative and quantitative analyses were performed using BioAnalyzer DNA 12000 kit (Agilent Technologies), and Fragment Analyzer Large-Fragment kit (Agilent Technologies). Pacific Biosciences stand-alone Excel template was followed for primer and polymerase addition/cleanup and sequence data for each library was generated using MagBead loading on three independent SMRTcells on the Sequel (Pacific Biosciences) at 50, 65, and 75 pM respectively. Movie collection time was set at 10 hours with a pre-extension time of 120 minutes.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Sequel |
| |
|
| Data processing |
All reads were mapped to hg38 using minimap2 (Li, Bioinformatics 2018) using the following parameters: -ax splice -uf --secondary=no -C5 Homo_sapiens.GRCh38.dna.primary_assembly.fa
The resulting bam files were then filtered for mapping quality and unique mapping using samtools view -F 256 -q 20,
Genome_build: hg38
Supplementary_files_format_and_content: Binary alignment map (bam) and corresponding index files filtered for mapping quality (MAPQ>20) and unique mapping (SAM flag = 256)
|
| |
|
| Submission date |
Apr 01, 2020 |
| Last update date |
Sep 03, 2020 |
| Contact name |
Stefan A Muljo |
| E-mail(s) |
[email protected]
|
| Organization name |
NIH/NIAID
|
| Street address |
10 CENTER DR RM 11N306
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL26180 |
| Series (2) |
| GSE147896 |
PacBio long-read RNA-sequencing to identify differentially expressed genes and repetitive elements between Wild Type RBM4 KO HAP1 cell line samples |
| GSE147897 |
Post-transcriptional regulation of human endogenous retroviruses by RNA-Binding Motif Protein 4, RBM4 |
|
| Relations |
| BioSample |
SAMN14517980 |
| SRA |
SRX8042049 |
