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

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Lab

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RPKM of yYH1

RNA-seq data on mRNA abundance agree well withpreviously published measurements

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Mock Rnase I

40s

60s80s

polysomes

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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…

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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