|
| Status |
Public on Aug 11, 2020 |
| Title |
NC1-Input |
| Sample type |
SRA |
| |
|
| Source name |
HCCLM3 cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HCCLM3 cells
cell type: hepatocellular carcinoma
genotype/variation: ALKBH5 -normal
merip antibody: none
|
| Treatment protocol |
HCCLM3 cells were transfected with lentiviruses expressing Flag-ALKBH5 (OE groups) or empty vector (NC groups) (MOI=30). They were then filtrated using 3-5 μg/ml puromycin for more than one week until the efficiency was examined by qPCR and immunoblot analysis.
|
| Growth protocol |
HCCLM3 cells were incubated at 37°C in 5% CO2 incubator with the humidified environment. And they were cultured with Minimum Essential Media (MEM) which were routinely supplemented with 10% fetal bovine serum (FBS).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with TRIzol reagent.
mRNA was enriched from total RNA with Poly(A) mRNA Magnetic Isolation Module(NEB#E7490) before fragmented chemically to ~150 nts long size, 10% fragmented mRNA was saved as Input sample while the rest part was subjected to IP using anti-m6A antibodies(Synaptic system#202 003). After stringent washing, bound RNA was eluted by competition with N6-methyladenosine (Sigma-Aldrich#M2780) as IP samples. IP and input library construction was carried out simultaneously with VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina (Box2&3, Vazyme#NR603). Qualities of each library were validated using an Agilent 2100 Bioanalyzer, and successful libraries of sample pairs (input and IP) were sequenced on illumina Novaseq sequencer with PE150 strategy.
MeRIP-seq
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence.
For meRIP seq, after removal of rRNA and tRNA, sequenced reads were mapped to hg19 using hisat2-align-s (v2.0.5). PCR duplicates were further removed with Picard (v2.16.0) and high quality mapped reads were extracted by samtools view (v0.1.09) “-q 30”. Differenct Regions enriched in the meRIP RNA samples over input RNA samples were identified as m6A peaks using exomePeak(v2.6.0) peak-caller.
Genome_build: hg19
Supplementary_files_format_and_content: Supplementary_files_format_and_content: bed files were generated using exomePeak.
|
| |
|
| Submission date |
Apr 28, 2020 |
| Last update date |
Aug 11, 2020 |
| Contact name |
Yunhao Chen |
| Organization name |
The First Affiliated Hospital, College of Medicine, Zhejiang University
|
| Street address |
79 Qingchun Road
|
| City |
Hangzhou |
| ZIP/Postal code |
310000 |
| Country |
China |
| |
|
| Platform ID |
GPL20301 |
| Series (1) |
| GSE149510 |
ALKBH5 impacts malignancy of hepatocellular carcinoma via m6A-mediated epigenetic modulation |
|
| Relations |
| BioSample |
SAMN14760492 |
| SRA |
SRX8190754 |
