|
| Status |
Public on Sep 03, 2020 |
| Title |
RBM4 KO Clone 1 replicate 3 |
| Sample type |
SRA |
| |
|
| Source name |
KBM-7 derived near-haploid cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HAP1
genotype: RBM4 KO
|
| 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 isolated from the HAP1 cells (WT and RBM4 KO) using the RNAzol®RT (Molecular Research Center) per manufacturer’s instruction. RNA was quantified with Qubit® RNA HS Assay Kit according to the manufacturer’s recommendation (Thermo Fisher Scientific). RNA integrity was verified with Agilent RNA 6000 Nano kit on the 2100 Bioanalyzer (Agilent Technologies) or HS RNA Kit (15 nt) on the Fragment Analyzer (Agilent Technologies) according to the manufacturers’ protocol.
RNA-Seq libraries were prepared from 65 ng of total RNA and 1 μL of 1:5000 (v/v) dilution of ERCC ExFold RNA Spike-In Mix 1 (Thermo Fisher Scientific) with TruSeq® Stranded mRNA Library Prep for NeoPrepTM instrument as per the NeoPrep Library Prep System Guide on the instrument (Illumina). Libraries were diluted 1:5 (v/v) and 1 μL was used to check the expected size (~300 bp) and the purify was verified with the Agilent High Sensitivity DNA Kit as manufacturer’s recommendation (Agilent Technologies). One μL of the diluted libraries were used to quantify as recommended in the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific) with standard range from 0 to 14 ng/ well. Libraries were normalized with molecular grade water to 2 nM with the above expected size and quantification. Pooled libraries of 2 nM were denatured and diluted as per the Standard Normalization Method for the NextSeq® System (Illumina). Final loading concentration of 1.8 pM and 1% PhiX (v/v) was used with the NextSeq® 500/550 High Output Kit v2 (150 cycles) (Illumina). Samples were sequenced paired-end with 75 cycles and single index of 6 cycles.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
1B2_3
|
| Data processing |
Reads were mapped to genome (hg38) using STAR (version 2.5.2a) (62), allowing for multi-mapping (parameters --outFilterMultimapNmax 100 --winAnchorMultimapNmax 100
STAR alignment BAM files were passed to the TEToolkit (Jin et al., Bioinformatics 2015), along with transcriptome and TE GTFs to be processed for gene and TE counts
TEToolkit parameters for paired-end samples: --mode uniq --GTF Homo_sapiens.GRCh38.85.gtf --TE GRCh38_rmsk_TE.gtf --strand reverse
TEToolkit parameters for single-end samples: --mode multi --stranded=no --GTF Homo_sapiens.GRCh38.85.gtf --TE GRCh38_rmsk_TE.gtf
Genome_build: hg38
Supplementary_files_format_and_content: Read count tables per sample/gene are provided separately for paired-end and single-end sample as tab-delimited text (.tsv) files.
|
| |
|
| 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 |
GPL18573 |
| Series (2) |
| GSE147893 |
RNA-sequencing experiments to identify differentially expressed genes between Wild Type and RBM4 KO HAP1 cell line samples |
| GSE147897 |
Post-transcriptional regulation of human endogenous retroviruses by RNA-Binding Motif Protein 4, RBM4 |
|
| Relations |
| BioSample |
SAMN14517971 |
| SRA |
SRX8042036 |
