RNAseq example
description
Kathi Zarnack data
Cell. 2013
Direct competition between hnRNP C and U2AF65 protects the transcriptome from the exonization of Alu elements.
Zarnack K, König J, Tajnik M, Martincorena I, Eustermann S, Stévant I, Reyes A, Anders S, Luscombe NM, Ule J.
https://www.ncbi.nlm.nih.gov/pmc/articles/
ALU elements and transposomes

An Alu element is a short stretch of DNA
originally characterized by the action of the
Arthrobacter luteus (Alu) restriction
endonuclease.

Alu elements are the most abundant
transposable elements (transposon,
jumping gene), containing over one million
copies dispersed throughout the human
genome.

Transposon is a DNA sequence that can
change its position within a genome, sometimes
creating or reversing mutations and altering the
Summary of the results
There are ~650,000 Alu elements in transcribed regions of the human genome. These elements
contain cryptic splice sites, so they are in constant danger of aberrant incorporation into
mature transcripts. Despite posing a major threat to transcriptome integrity, little is known about
the molecular mechanisms preventing their inclusion.
Here, we present a mechanism for protecting the human transcriptome from the aberrant
exonization of transposable elements. Quantitative iCLIP data show that the RNA-binding protein
hnRNP C competes with the splicing factor U2AF65 at many genuine and cryptic splice sites.
Loss of hnRNP C leads to formation of previously suppressed Alu exons, which severely disrupt
transcript function. Minigene experiments explain disease-associated mutations in Alu elements
that hamper hnRNP C binding. Thus, by preventing U2AF65 binding to Alu elements, hnRNP C plays a
critical role as a genome-wide sentinel protecting the transcriptome. The findings have
important implications for human evolution and disease.
Cell. 2013
Direct competition between hnRNP C and U2AF65 protects the transcriptome from the exonization of Alu elements.
Zarnack K, König J, Tajnik M, Martincorena I, Eustermann S, Stévant I, Reyes A, Anders S, Luscombe NM, Ule J.
Design of the experiment

The aim was to find out what the HNRNPC gene does

The experiment performed a knockdown of the HNRNPC gene in HeLa cells
(hnRNP C Stealth Select RNAi siRNAs HSS179304 and HSS179305) as
well as control siRNA Stealth RNAi siRNA Negative Control (Invitrogen).
 SiRNA negative control (control) – 2 samples
 KD1 – knock-down 1 – 2 samples
 KD2 – knock-down 2 – 2 samples
 RNA-seq libraries were sequenced on an Illumina GA-2 (72 cycles,
paired end)


HeLa

is an immortal cell line used in scientific research. It is the oldest and most
commonly used human cell line. The line is derived from cervical cancer cells
taken on February 8, 1951,from Henrietta Lacks, a 31-year-old AfricanAmerican
mother of five, who died of cancer on October 4, 1951. The cell
line was found to be remarkably durable and prolific, which allows it to be
used extensively in scientific study.
Design of the experiment

The experiment performed a knockdown of the HNRNPC gene in HeLa cells
(hnRNP C Stealth Select RNAi siRNAs HSS179304 and HSS179305) as
well as control siRNA Stealth RNAi siRNA Negative Control (Invitrogen).
 SiRNA negative control (control) – 2 samples
 KD1 – knock-down 1 – 2 samples
 KD2 – knock-down 2 – 2 samples
 RNA-seq libraries were sequenced on an Illumina GA-2 (72 cycles,
paired end)

Goal of the practical
Get from the raw sequencing data to the gene expression (RNA-Seq)
Analyze RNA-Seq data and get differential gene expression and
expression of individual exons (example at gene CD55 gene)
Show coverage cryptic exon(s) (example at gene CD55)