Ribosome Profiling Library Preparation with SOLID Nate Blewett MGL Users Group May 4 th 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 i 6 A adjacent to the anticodon

m7Gm7G AAAAAA Rnase I Ribosome Protected fragments Gel purification of RPFs Dephosphorylate 3' Phosphorylate 5' Adaptor Ligation Reverse Transcription & PCR Library building Analysis of global translation via ribosome profiling 7% 47% m7Gm7G AAAAAA m7Gm7G 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 Bahler Lab microarray RPKM of yYH1 RNA-seq data on mRNA abundance agree well with previously published measurements Marguerat, et al., Cell 2012

Mock Rnase I 40s 60s 80s 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 RPF 3' 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…

unligated marker RT ligated marker RT Gel slices are excised and PCR performed on slices using 8 and 12 cycles 8 cycles 12 cycles bp PCR libraries from the lowest cycle number to give good product are gel-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 Lab Rich Maraia Sergei Gaidamakov Sandy Mattijssen Aneesh Arimbasseri Vera Cherkasova Keshab Rijal Matt Smith Thanks! MGL Steve Coon James Iben