Ribosome Profiling Library Preparation with SOLID Nate Blewett MGL Users Group May 4 th 2015.
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Transcript of Ribosome Profiling Library Preparation with SOLID Nate Blewett MGL Users Group May 4 th 2015.
Ribosome Profiling Library Preparation with SOLID
Nate Blewett
MGL Users Group
May 4th 2015
• i6A modification of A37 adjacent to anticodon modulates the ability of tRNAs to decode certain codons
• We hope to utilize ribosome profiling to understand what impact i6A tRNA modification has on ribosome decoding activity/pausing and identify mRNAs that are poorly translated upon loss of i6A tRNA modification
TRIT1/Tit1 modifies subsets of tRNAs with i6A adjacent to the anticodon
m7G
AAAAAA
Rnase I
RibosomeProtectedfragments
Gel purification of RPFs
Dephosphorylate 3'Phosphorylate 5'
Adaptor Ligation
Reverse Transcription & PCRLibrary building
Analysis of global translation via ribosome profiling
7%
47%
m7G
AAAAAA
m7G
AAAAAA
Fractionation of digested ribosome protected fragments
Samples re-extracted
Ribosome protected Fragment Reads are normalized to mRNA abundance quantified via RNA-seq
rRNA removal validation
• RNA samples were Dnase treated and rRNA depleted
• RNA-seq libraries were prepared and sequenced by the MGL with thanks to James Iben and Steve Coon
r = 0.74
Bah
ler
Lab
mic
roar
ray
RPKM of yYH1
RNA-seq data on mRNA abundance agree well withpreviously published measurements
Mar
guer
at, e
t al.,
Cel
l 201
2
Mock Rnase I
40s
60s80s
polysomes
80s
Optimization of Ribosome Profiling Library Preparation for the SOLID Sequencing Platform
• All published ribosome profiling has been performed on the Illumina platform.
• Regardless of platform, high-quality, intact polysome isolation is crucial for accurate reporting on translation
• Vacuum harvesting followed by mortar and pestle lysis under LN2
• Lysates are digested to destroy everything but ribosome protected mRNA fragments
• The number of reads relative to ORF length determines the relative ribosome occupancy for each mRNA
Gel purification of Ribosome Protected Fragments
• Overnight gel-extraction, followed by precipitation
• rRNA was depleted from gel-purified 26-34nt fragments
• 3' end is then dephosphorylated with T4 PnK
Linker Ligation Protocol from Life Tech. Benefited from Optimization
RPF3' linker5' linker
DNA guide
• The linkers have nothing blocking their 3' end, allowing them take part in several off-target undesired ligation events
• The 5' and 3' ligations are supposed to be performed simultaneously which increases the possibilities for unproductive ligations
• T4 RNA ligase will ligate RNA:DNA molecules which further increases the possibility for issues.
no DNA guide DNA guide
The 5' end of the RPFs is then phosphorylated, and ligated to a 5' adaptor
Four libraries were then prepared and sequenced…
unlig
ated
mar
ker
RT
ligat
ed m
arke
r R
TGel slices are excised and PCR performed on slices using 8 and 12 cycles
8 cycles 12 cycles
120-140 bp
PCR libraries fromthe lowest cycle number to give good product aregel-purified
5' and 3' Ligated RPFs are Reverse Transcribed, then Libraries are PCR Amplified
rRNA Contamination Required Optimization
• Libraries came back with 80% of the reads mapping to rRNA sequences
• This is the same level as Ingolia reported for his first ribo-seq experiment which didn't have a step for rRNA removal
• The rRNA removal kit targets a limited number of sequences that is effective if the RNA is intact.
• There are two predominant rRNA species present that are roughly the size of the RPFs, and are not targeted by the RiboZero kit
• Based on the SOLID sequencing data we had two biotinylated oligos synthesized to target the contaminating rRNA species.
• 4 libraries were prepared again and submitted for sequencing, which showed that roughly 50% of reads were rRNA. The majority of published ribosome profiling studies report very similar levels of rRNA contamination
Maraia LabRich MaraiaSergei GaidamakovSandy MattijssenAneesh ArimbasseriVera CherkasovaKeshab RijalMatt Smith
Thanks!
MGLSteve CoonJames Iben