|Public on Jul 25, 2019
|Wild-type replicate 2 for RNA-seq
|cell line: HEK293T
|6.4x10^6 cells of HEK293T WT or XRN1-null cells were plated on 15 cm dishes 24 hrs before harvesting.
|Total RNA was extracted using the RNeasy Mini Kit (Qiagen) and a library prepared using the TruSeq RNA Sample Prep Kit (Illumina). For Ribo-Seq, the original procedure of Ingolia et al (2012) Nature Protocols was followed with modifications from Calviello et al (2016) Nature Methods.
RNA libraries were prepared for sequencing using standard Illumina protocols
|Illumina HiSeq 3000
|Illumina software used for basecalling.
Adapters removed with Flexbar, ribosomal-derived reads were removed using Bowtie2
Remaining reads were then mapped on hg19 using Tophat2
Ribo-seq reads were analyzed for three-nucleotide periodicity using the RiboTaper
Read count analysis was done with an R/Bioconductor package QuasR
Differential gene expression analysis with an R/Bioconductor package edgeR
Translational efficiency (TE) was estimated using the statistical framework and analysis as implemented in the RiboDiff program
Supplementary_files_format_and_content: Tab-delimited file for readcounts; tab-delimited file for differential expression analysis; tab-delimtied file for translational efficiency analysis
|Jun 13, 2019
|Last update date
|Jul 25, 2019
|Max Planck Institute for Developmental Biology
|Transcriptome and ribosome profiling of XRN1-null HEK293T human cell line generated by CRISPR/Cas9 gene editing