Construction of High-Quality Rice Ribosome Footprint Library

Abstract

High-throughput sequencing of ribosome footprints precisely maps and quantifies in vivo mRNA translation. The ribosome footprint sequencing has undergone continuing development since its original report. Here we provide a detailed protocol for construction of high-quality ribosome footprint library of rice. Rice total polysomes are isolated with a modified low ionic polysome extraction buffer. After nuclease digestion, rice ribosome footprints are extracted using SDS method followed by column purification. High-quality rice ribosome footprint library with peak reads of approximately 28-nucleotide (nt) length and strong 3-nt periodicity is constructed via key steps including rRNA depletion, end repair, 3' adapter ligation, reverse transcription, circularization, PCR enrichment and several rounds of purification. Biological significance of rice ribosome footprint library is further revealed by the comparison of transcriptomic and translatomic responses to salt stress and the utilization for novel open reading frame (ORF) identification. This improved protocol for rice ribosome footprint library construction will facilitate the global comprehension and quantitative measurement of dynamic translation in rice.

Ribosome profiling reveals the translational landscape and allele-specific translation efficiency in rice

“…However, in rice, we observed that the vast majority of RPFs are 26–28nt in length, particularly RPFs of 27nt (Figure 1B). This unique feature was also observed in a previous study, in which a high-quality rice ribosome footprint library was constructed (Yang et al ., 2020). In total, 96.0% of RPFs could be mapped to annotated coding region (CDS), while the remaining RPFs were almost equally distributed in 5’UTR, 3’URT and intron (Figure 1C).…”

Comparative ribosome profiling reveals distinct translational landscapes of salt-sensitive and -tolerant rice

“…Polysomes were isolated by the method described by Yang et al [ 73 ]. Briefly, about 1 g of pulverized NB or SR86 shoots in liquid nitrogen was transferred to a 15-mL nuclease-free centrifuge tube with 5 mL ice-cold polysome extraction buffer (PEB) [100 mM Tris-HCl (pH 8.0), 40 mM KCl, 20 mM MgCl 2 , 2% (V/V) polyoxyethylene-10-tridecyl ether, 0.2% (W/V) deoxycholic acid, 1 mM DTT, 100 μg mL − 1 cycloheximide and 10 U mL − 1 DNaseI] and mixed well.…”

“…The filtrate was subjected to rRNA removal with the Ribo-Zero rRNA Removal Kit for Plant Leaf (MRZPL1224, ILLUMINA, USA). Finally, RPFs around 28–30 nt were recovered by PAGE purification as described previously [ 73 ].…”

“…Ribo-seq libraries for NB and SR86 were constructed with the recovered RPFs, and then subjected to PCR enrichment with TruSeq Ribo Profile Forward and Index primers, according to previously described method [ 73 ]. The PCR products were purified with AMPure XP beads (BECKMAN, USA), followed by 8% (W/V) native PAGE.…”

“…The PCR products were purified with AMPure XP beads (BECKMAN, USA), followed by 8% (W/V) native PAGE. The bands ranging from 140 to 160 bp were excised from the native PAGE and libraries were recovered from the excised gel pieces by overnight elution [ 73 ].…”