Isogen 2 reagent

Human embryonic kidney 293 (HEK293) (JCRB9068, JCRB Cell Bank) and TIG-1 (spontaneously developed diploid fibroblast cell lines of fetal lung [JCRB0501, JCRB Cell Bank]) cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Nacalai Tesque) with 10% supplemented with fetal bovine serum (Gibco). Human primary lung (PCS-201-013, ATCC), adult dermal (PCS-201-012, ATCC) and neonatal dermal cells (PCS-201-010, ATCC) were cultured in the recommended media. Cells were maintained at 37°C in a humidified chamber supplemented with 5% CO₂. For tRNAseq and northern blot, 3.0–6.0 × 10⁶ cells were seeded on 100 mm dishes using a culture medium. For i-tRAP and RT-qPCR, 4.0–8.0 × 10⁵ cells were seeded on six well plates.
For amino acid starvation, cells were cultured in amino acid­–free DMEM (Wako) supplemented with 0.5% dialyzed fetal bovine serum (Gibco) for indicated time. Amino acid supplementation was conducted using MEM essential amino acids solution, MEM nonessential amino acids solution, and 200 mM glutamine solution (Wako) for the indicated time.
Small RNA was extracted from cells using Isogen II reagent (Nippongene) and Ethachinmate (Nippongene) according to manufacturer's instruction.

Total RNA from cell lines was prepared using ISOGEN II reagent (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. The total RNA samples were treated with recombinant DNase I (Takara Bio, Kusatsu, Japan) and then reverse‐transcribed to single stranded cDNA using ReverTra Ace reverse transcriptase (Toyobo, Osaka, Japan) with random primers in accordance with the manufacturer's instructions. The cDNA was amplified using THUNDERBIRD SYBR qPCR Mix (Toyobo) or TaqMan Fast Universal PCR Master Mix (Life Technologies, Carlsbad, CA, USA) and quantified using a StepOnePlus Real‐Time PCR System (Life Technologies). β‐actin (Life Technologies, #4326315E) was used for normalization.