|Public on Jun 30, 2021
|cervix adenocarcinoma cell line
|cell line: HeLa
cell type: cervix adenocarcinoma cell line
genotype: lentiviruses expressing blank vector
|These cells were cultured in DMEM medium supplemented with 10% fetal bovine serum at 37℃ in a humidified incubator with 5% CO2.
|Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure. mRNA was purified using poly-T oligo attached magnetic beads (Invitrogen). mRNA was then fragmented into ~100nt oligonucleotides using divalent cations under elevated temperature. Fragmented mRNA was incubated with m6A-specific antibody in IP buffer (50 mM Tris-HCl, 750 mM NaCl and 0.5% Igepal CA-630) supplemented with 0.5 μg/μl BSA for 2h at 4 ℃. Immunoprecipitated m6A RNA fragments were used for the construction of m6A-seq libraries (IP). And 1 ug of total RNA was used for the construction of RNA-sequencing libraries (Input).
RNA libraries were prepared for sequencing using standard Illumina protocols.
Immunoprecipitated m6A RNA fragments for m6A-seq (IP), total RNA for RNA-Seq (Input).
|Illumina NovaSeq 6000
|Cutadapt and perl scripts in house were used to remove the reads that contained adaptor contamination, low quality bases and undetermined bases. Then sequence quality was verified using FastQC.
All reads were mapped to human genome by bowtie with default settings.
Mapped reads of m6A fragments and input libraries were provided through R package exomePeak, which identifies m6A peaks with bed or bam format that can be adapted for visualization on the UCSC genome browser or IGV software (http://www.igv.org/).
Called peaks were annotated by intersection with gene architecture using ChIPseeker. Then StringTie was used to perform expression level for all mRNAs from input libraries by calculating FPKM.
Genome_build: Version GRCh38.p10
|Dec 17, 2019
|Last update date
|Jun 30, 2021
|Biomedicine Research Center, the Third Affiliated Hospital of Guangzhou Medical University
|63 Duobao Road
|ALKBH5 K235 acetylation regulates the RNA m6A profiles