|
| Status |
Public on Jun 01, 2023 |
| Title |
Strep-ALKBH5 Rep 1 (RIP-seq) |
| Sample type |
SRA |
| |
|
| Source name |
Head&Neck SCC cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: UM-SCC-1
cell type: Head and neck squamous cell carcinoma (HNSC)
treatment: StrepTactin-XT RIP
tag: Strep-ALKBH5
|
| Treatment protocol |
For the RNA-Seq and m6A-Seq assay, UM-SCC-1 cells were infected with si RNA scramble, ALKBH5 siRNA#1/2, RBM33 siRNA#1/#2 retrovirus and selected with10 ug/mL blasticidin for 4 days. For the RIP-Seq assay, UM-SCC-1 cells were infected with strep-Vec, strep-ALKBH5, and strep-RBM33 lenti-virus and selected with 1 ug/mL puromycin for 4 days. Then infect UM-SCC-1 cells stably expressing strep-ALKBH5 with RBM33 siRNA#1/#2 retrovirus and selected with blasticidin for another 4 days.
|
| Growth protocol |
UM-SCC-1 cells were rountinely cultured in 10 cm dishes with DMEM mediun supplemented with p.s and FBS and cell passge was pefromed by 0.25% trypsin digestion when cell conflunece reaches 95%.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Lysates were obtained from lysis buffer containing 0.1% NP-40. RNA was purified and fragmented with the BioRuptor Pico.
Libraries were prepared at half scale according to the manufacturer's instructions accompanying the Takara SMARTER Stranded Total RNA-seq Kit v2 - Pico Input Mammalian (Cat # 634412). Libraries were then inspected with the Bioanalyzer machine. Libraries were sequencied on the Illumina NovaSeq 6000 according to manufacturer's instructions.
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Adapters of all raw RIP-seq data sets were removed by Cutadapt v1.18.
Then reads longer than 19 nt were mapped to the human genome (hg38) and the GENCODE v36 gene annotation using HISAT2 v2.1.0 with default parameters.
Raw reads on each gene were counted by featureCounts (-t exon -g gene_id -M -O --fraction -T 24 -s 2) from Subread v1.6.4.
For each gene, the differential enrichment of reads between IP and input samples were calculated by the edgeR v3.24.3 package.
Genes with the cutoff (log2FCIP/input > 1 and FDR < 0.05) were determined as candidate targets and used for further analysis.
genome_build: hg38
Supplementary_files_format_and_content: Fold changes of read abundance and p-values between IP and input groups in the plain txt format
|
| |
|
| Submission date |
May 12, 2022 |
| Last update date |
Jun 01, 2023 |
| Contact name |
Allen Zhu |
| E-mail(s) |
allenczhu@uchicago.edu
|
| Phone |
7738343468
|
| Organization name |
University of Chicago
|
| Street address |
929 E 57th St, Room E331
|
| City |
Chicago |
| State/province |
Illinois |
| ZIP/Postal code |
60637 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (2) |
| GSE202878 |
RBM33 Acts as an m6A Reader Essential for ALKBH5-mediated m6A Demethylation and Head and Neck Squamous Carcinoma Tumorigenesis |
| GSE242281 |
RBM33 Acts as an m6A Reader Essential for ALKBH5-mediated m6A Demethylation and Head and Neck Squamous Carcinoma Tumorigenesis [RIP-seq] |
|
| Relations |
| BioSample |
SAMN28206695 |
| SRA |
SRX15243998 |
