|
| Status |
Public on Jun 28, 2022 |
| Title |
Input_ALKBH5_Rep3 |
| Sample type |
SRA |
| |
|
| Source name |
Osteosarcoma
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: 143B
cell type: Osteosarcoma
sample type: fragmented input RNA
treatment: ALKBH5 knockdown
harvest time: 48 hour
rip antibody: none (input)
|
| Treatment protocol |
143B cells were grown in 150 mm tissue culture plates and transfected with with either scrambled-siRNA or ALKBH5-siRNAs (Sigma-Aldrich) for 48 hours. Transfection was carried out using the RNAiMAX Reagent (Thermofisher) according to the manufacturer’s protocol.
|
| Growth protocol |
143B cells were cultured in minimum essential medium (Eagle) supplemented with 10% fetal bovine serum and 1% penicillin/streptamycin. Cells were maintained in a humidified incubator at 37°C and 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from scrambled and ALKBH5-siRNA transfected 143B cells using Trizol (Thermo 15596018) followed by mRNA isolation using a Dynabeast Direct Kit (Life Tech 61012). 2 μg of polyadenylated mRNA was diluted to 100 ml in water and fragmented to 100 to 200 nucleotides using Bioruptor (Diagenode). 5 ml of RNA was used for input preparation. 2 μg of fragmented mRNA was used for immunoprecipitation (IP) using rabbit anti-m6A antibody (Synaptic Systems #202003) in IP buffer (10 mM Tris pH 7.4, 150 mM NaCl, 0.1% NP-40) for 2 hours at 4°C. After incubation, samples were added to Protein A beads and incubated for 2 more hours at 4°C with rotation. After washing, samples were eluted using elution buffer containing m6A nucleoside and elute was concentrated using a Zymo RCC-5 column. The resultant final product was used for RNA-seq. 0.1 μg of fragmented mRNA was used as input control for RNA-seq. For replicate 3: same protocol was followed with another m6A antibodiy (ABE572, Millipore, Germany). .For rep2, meRIP-seq was done with total RNA using the Magna MeRIP m6A kit (catalog no.17-10499, Millipore) according to the manufacture’s protocol and it employed MABE1006 (Millipore-Sigma) m6a antibody for IP.
RNA-seq library preparation by following the Illumina TruSeq stranded mRNA sample preparation guide
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 3000 |
| |
|
| Data processing |
Illumina Casava v1.8.2 software used for base-calling
All RNA-seq FastQ reads were aligned with the reference genome (UCSC human genome build hg19) using TopHat2 default settings. The aligned BAM files sorted (SAMTools) and then processed to remove reads with low alignment quality.
BAM files were converted to TDF format using IGVTools for visualization. The m6A enrichment peaks were called using MeTPeak package.
Genome_build: hg19
Supplementary_files_format_and_content: The processed MePeak files are 12-column BED format with column header. The tiled data files (.tdf or TDF) are binary files that contain data that has been preprocessed for faster display in IGV
|
| |
|
| Submission date |
Jul 15, 2020 |
| Last update date |
Jun 28, 2022 |
| Contact name |
Yidong Chen |
| E-mail(s) |
cheny8@uthscsa.edu
|
| Phone |
2105629163
|
| Organization name |
UT Health Science Center at San Antonio
|
| Department |
Population Health Sciences
|
| Street address |
8403 Floyd Curl Drive, MSC 7784
|
| City |
San Antonio |
| State/province |
Texas |
| ZIP/Postal code |
78229 |
| Country |
USA |
| |
|
| Platform ID |
GPL21290 |
| Series (2) |
| GSE154529 |
Identification of ALKBH5 targets in osteosarcoma by methyl RNA immunoprecipitation [MeRIP-seq] |
| GSE154530 |
Gene regulation by m6A demethylase ALKBH5 in osteosarcoma |
|
| Relations |
| BioSample |
SAMN15546733 |
| SRA |
SRX8741396 |
