Computational protocol: Replication fork integrity and intra-S phase checkpoint suppress gene amplification

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Protocol publication

[…] Total RNA was isolated from Mre11-KD and control cells (infected with LV-GFP) using RNeasy plus mini kit (QIAGEN). Two microgram of total RNA was processed to construct sequencing libraries using TrueSeq RNA Sample Preparation Kit (Illumina) at the Genomics Core at Cleveland Clinic Lerner Research Institute. Thirty-five base pair, single-end sequencing was done using Genome Analyzer IIx at the Nucleic Acid Shared Resource of Ohio State University. Library construction and sequencing were performed in duplicate for Mre11-KD cells and control cells. The numbers of reads obtained were: 24 504 094 (control-1), 26 689 524 (control-2), 27 404 397 (Mre11KD-1) and 26 288 201 (Mre11KD-2). GEO accession number for the sequences is GSE59487.Fastq files containing RNAseq data were aligned to the Chinese hamster reference genome (July 2013 (C_griseus_v1.0/criGri1) assembly downloaded from the UCSC database) using Tophat. Gene scaffold data (ref_CriGri_1.0_scaffolds.gff3) was included in the alignment. After alignment, the total number of reads mapping to each exon was counted using custom perl scripts. The edgeR package within R was used to quantify gene expression differences between Mre11-KO and control cells (). To calculate false discovery rates, we used the q-value R package, which estimates the proportion of false positives based on the overall distribution of P-values. […]

Pipeline specifications

Software tools TopHat, edgeR
Application RNA-seq analysis
Organisms Homo sapiens