library_construction_protocol: Cells grown in 10 cm dishes were washed with 5 mL of ice-cold PBS containing 100 μg/mL cycloheximide (Millipore, Cat. #239764) and immediately harvested by scraping. The cells were collected by centrifugation at 5,000 × g for 5 minutes, and the pellet was resuspended in 400 μL of lysis buffer. The lysate was homogenized by vortexing and incubated on ice for 10 minutes. Clarification was performed by centrifugation at 15,000 × g for 10 minutes at 4°C, and the supernatant was kept on ice. For RNA digestion, 40–60 μg of RNA from the lysate were treated with RNAse I (Lucigen, Cat. #N6901K) for 50 minutes at room temperature while rotating. Following digestion, 5 μL of RNasin™ Plus RNAse inhibitor (Promega, Cat. #PRN2615) was added to the RNA. Monosome isolation was performed with Microspin S-400 HR columns (Cytiva, Cat. #GE27-5140-01). Columns were washed six times with 500 μL of column wash buffer, followed by centrifugation at 600 × g for 4 minutes. Subsequently, 100 μL of digested sample was applied to the resin in each column and centrifuged at 600 × g for 2 minutes. Approximately 125 μL of eluate was collected per column, and eluates from the same sample were pooled. To precipitate Ribosome-Protected Fragment (RPF) RNA, an equal volume of acid phenol:chloroform was added to the eluate, vortexed, and incubated at room temperature for 5 minutes. The mixture was centrifuged at 15,000 × g for 10 minutes at 4°C. The aqueous phase was collected, and 1:10 volume of 3 M NaOAc (pH 5.2), 2 μL of glycogen, and 1 volume of ice-cold isopropanol were added. The RPF RNA was precipitated overnight at −80°C. The samples were centrifuged at 15,000 × g for 15 minutes at 4°C, the supernatant was discarded, and the pellet was washed with 80% ethanol. Following another centrifugation at 15,000 × g for 10 minutes at 4°C, the pellet was air-dried and resuspended in 15 μL of nuclease-free water. To select for the appropriate size RNA, the samples were run on a Novex 12% TBE Urea precast gel (Thermo Fisher Scientific). 5uL of Gel Loading Buffer (Thermo Fisher, Cat # AM8547) was added to the resuspended RNA and loaded into one or two wells of the gel alongside a 30nt RNA oligo marker and small RNA ladder (Zymo Cat # R1090). The gel was then washed with TBE, stained with Sybr Gold Nucleic Acid Stain (Thermo Fisher, Cat. #S11494), and imaged. RNA species of 30nt using the marker as reference were then excised, crushed, and suspended in 400uL RNA Binding Buffer (Zymo RNA Clean & Concentrator Kit, Cat. # R1016) and left to diffuse at room temperature overnight. Following overnight diffusion, samples were centrifuged at 10,000 x g for 10min to pellet the gel and ~350uL of RNA binding buffer was removed. The RNA was then isolated using the RNA Clean & Concentrator™ column (Zymo Research), following the manufacturer's instructions and eluted in 30uL of nuclease free water. Following elution, samples were transferred to PCR tubes, mixed with 1uL (10 units) of T4 Polynucleotide Kinase (NEB, Cat #M0201L), 5uL T4 Polynucleotide Kinase Reaction Buffer (NEB, Cat #B0201S) and 5uL 10mM ATP, and the volume was brought up to 50uL. Samples were then incubated at 37C for 1 hour. A modified protocol for the RNA Clean & Concentrator™ column was then used to isolate RNA. 100uL adjusted binding buffer (1:1 RNA Binding buffer and ethanol) was mixed with the phosphorylated RNA and added to the column. After centrifugation, 150uL of ethanol was then added to the flow through and this solution was loaded added to a new column and washed according to the manufacturer's instructions, eluting in 7uL of nuclease free water. Library preparation was performed using the Takara SMARTer® smRNA-Seq Kit for Illumina® (Takara). Samples received 17 PCR cycles and were sequenced at 50-bp single end reads on a NovaSeq6000