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RNA sequencing and itsapplicationsAndrew Tolonenatolonen "at" genoscope.cns.fr@andrew_tolonenhttp://www.tolonenlab.org
Deplete ribosomal RNA (rRNA)
Illumina sequencing
dRNA-seq captures transcription start sites
Filiatrault 2011 PMID 22216251
TEX degrades RNA with 5' phosphate
TEX=5' monophosphate dependent terminator exonuclease
Fig B Helicobacter pylori "HP" and Salmonella typhimurium "ST" without "-"or with "+" TEX treatment.
TEX degrades RNA with 5' P (rRNA,tRNA)
PMID 20164839
dRNA-seq defines promoters (5' UTR)
PMID 20164839
Most H. pylori promoters are 20-40 bp
ribosome binding site (AAGGA/G)
26 genes haveno promoters
TAP converts 5' PPP on mRNAto 5' P
sodium periodate blocks 3' OH (no circularization)
make cDNA withrandom 3' primer
mRNA have 5' PPPrRNA, tRNA, degraded RNA have 5' P
ligate 5' RNA linker
remove rRNA, tRNA, partially degraded mRNA
dRNA-seq enrichment of mRNA TSS
increased TSS coveragein mRNA (5' PPP)
PMID 20164839
no change TSS coveragein tRNA (5' P)
RNase P is ribozyme that cleaves 5' leader from tRNA
-10 Pribnow box
AT-rich stretch
+TEX,TAP biases read distribution to 5'
yellow=5' UTRTSS=green arrow
*enrichment of 5' PPP RNA (all bacterial primary transcripts have 5' PPP)
dRNA-seq: antisense transcription in 46% H. pylori ORFs
HPnc1200 is antisense RNA
confirmation of antisenseRNA by Northern blot
PMID 20164839
dRNA-seq: discovery of 'hundreds' of candidate small RNA (sRNA) in H. pylori
candidate small RNA
60 sRNA validated by Northern blot
PMID 20164839
*gene calling algorithms often reject all genes <100 bp
sRNA regulate other genes by binding 5' UTR
Smart-seq single cell transcriptomics
PMID 24385147
RNA-seq is done on cell populations, but individual cells differ in gene expression
poly dT primers RT
RT adds a few 3' C's
add oligo with 3' G's to prime 2nd strand synthesis
Locked nucleic acid (LNA)
methyl bridge connects 2' O and 4' C
http://en.wikipedia.org/wiki/Locked_nucleic_acid
increased thermal stability of nucleic acid duplexes->shorter primers with higher specifity
make full length cDNA with 5' and 3' adapters
Template switching (TS)
TSO has 3' LNA G (red) binds with higher affinity
single primer PCR (18 cycles) for 1 ng cDNA
Tn5 Tagmentation
Illumina.com
transposase catalyzes in vitro DNA fragmentation and adaptor incorporation
hyperactive derivative of the Tn5 transposase catalyzes in vitro integration of syntheticoligonucleotides into target DNA
PCR to add library adapters
PCR and sequencing (96 multiplex)
Smart-seq summary
Advantages-analyze 96 single cell transcriptomes (ie regions of a tumor)-template-switching produces full-length cDNADisadvantages-requires polyT tails-not strand-specific
RNA sequencing is a method to measure mRNAexpression of all genes in the cell (transcriptomics)
http://www.cea.fr/presse/liste-des-communiques/decouverte-de-nouveaux-cocktails-enzymatiques-144237
we recently used RNA-seq to find enzymes that break apart plant biomass
purify mRNA from culture for RNA-seq
remove rRNA using oligos matching rRNA attached to magnetic beads
depletion of 23S (2904 bp) and 16S (1542 bp) bands
convert RNA into double stranded cDNA
don't know which strandmRNA came from
methods for strand-specific RNA-seq
ligate adapters to mRNA
RT templateswitching
each method has positives and negatives
probe with labeled copy of RNA
standard Illumina library preparation and sequencing
ligate y-shaped adapters, no concatamers
3' A tailing
produce cDNA in both orientations
phosphorothioate nucleotides are nuclease resistant
http://en.wikipedia.org/
replacment of an oxygen in the phosphate backbone with sulfurincreases nuclease resistance
dUTP sequencing library preparation
replace thymine with uracil in first strand synthesis
treat with USER (Uracil DNA glycosylase and Endonuclease VIII) to cut at uracils
PCR produces only 1 orientation of product
RNA-seq reads are highly strand-specific
Boutard et al, 2014
Today we will focus on 3 applications in RNA sequencing 1 strand-specificity 2 promoter mapping 3 measuring expression in single cells