Computational protocol: SPOP-containing complex regulates SETD2 stability and H3K36me3-coupled alternative splicing

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Protocol publication

[…] A total of 10 μg of RNA mixture was used for mRNA purification and RNA-Seq library preparation. Quality control was evaluated on Agilent 2100 and sequencing was performed on Illumina Hiseq4000 platform with 150 paired end mode. Approximately 90 million reads were generated for each sample, and the data presented were based on two independent RNA-seq experiments.Quality control for raw sequencing data was performed by FastQC v0.10.1. Low quality reads and adaptor contamination were removed by Cutadapt 1.8.3. After quality control and data filtering, reads were aligned to the reference genome hg19 by TopHat v2.0.10, allowing 2 mismatches per read and only concordant paired end reads were accepted for downstream analysis. MATS.3.0.8 beta and rmats2sashimiplot were used for alternative splicing analysis and related significant splicing events plot. Splicing events were accepted as significant with FDR < 0.05. Functional analysis and Gene Ontology (GO) analysis were performed by DAVID. GO plot R package was used for GO analysis plot and R version 3.2.2 was used for some custom analysis. [...] ChIP-Seq was performed by using Rubicon ThruPLEX DNA-seq kit according to manufacturer's instructions. Briefly, ChIPed DNA and matched input DNA were prepared for end repair and ‘A’ tailing, adaptor ligation and library amplification. ChIP-Seq sequencing was performed on Illumina HiSeq2500 platform with 50 bp single end sequencing. The data presented were based on two independent experiments.Quality control was performed with FastQC. CutAdapt was next used to trim low quality bases and adaptor sequences. Bowtie was used for data mapping to the hg19 reference genome, allowing two mismatches. Samtools was used to remove PCR-duplicated reads and only unique mapped reads were kept for downstream analysis. ChIP-Seq peaks calling was performed by MACS with P-value 1e-6. Genes that showed altered H3K36me3 levels were identified after normalizing all data to reads per million. The line plot of ChIP-Seq enrichment was performed by ngs.plot. […]

Pipeline specifications

Software tools FastQC, cutadapt, Bowtie, SAMtools, ngs.plot
Application ChIP-seq analysis
Diseases Neoplasms