Total RNA was isolated from HEK293T and HeLa cells using TRIzol (Invitrogen) and mixed with 1 μl of 10 nM spike-in control oligos, thirty non-human RNA sequences of 21–23 nt in length (Supplementary Table S1). The oligos were obtained from Bioneer Inc., resuspended in distilled water and pooled at equimolar concentrations. The RNA mixture was size-fractionated by 15% urea–polyacrylamide gel electrophoresis and eluted in 0.3 M NaCl to enrich miRNA species using two FAM-labeled markers (17 nt and 29 nt). Small RNA libraries were constructed using either the TruSeq small RNA library preparation kit (Illumina) according to manufacturer's instructions or AQ-seq as follows: miRNA-enriched RNA was ligated to 0.25 μM 3′ randomized adapter using 20 units/μl of T4 RNA ligase 2 truncated KQ (NEB) in 1X T4 RNA ligase reaction buffer (NEB) supplemented with 20% PEG 8000 (NEB) at 25°C for at least 3 h. The ligated RNA was gel-purified on a 15% urea–polyacrylamide gel using two markers (40 nt and 55 nt) to remove the free 3′ adapter and eluted in 0.3 M NaCl. The purified RNA was ligated to 0.18 μM 5′ randomized adapter using 1 unit/μl of T4 RNA ligase 1 (NEB) in 1X T4 RNA ligase reaction buffer supplemented with 1 mM ATP and 20% PEG 8000 (NEB) at 37°C for 1 h. The products were reverse-transcribed using 10 units/μl of SuperScript III reverse transcriptase (Invitrogen) in 1X first-strand buffer (Invitrogen) with 0.2 μM RT primer (RTP, TruSeq kit; Illumina), 0.5 mM dNTP (TruSeq kit; Illumina), and 5 mM DTT (Invitrogen) at 50°C for 1 h. The cDNA was amplified using 0.02 unit/μl of Phusion High-Fidelity DNA Polymerase (Thermo Scientific) in 1X Phusion HF buffer (Thermo Scientific) with 0.5 μM primers (RP1 forward primer and RPIX reverse primer, TruSeq kit; Illumina) and 0.2 mM dNTP (TAKARA). The PCR-amplified cDNA was gel-purified using a 6% polyacrylamide gel to remove adapter dimers and sequenced using MiSeq or HiSeq platforms. Markers for size-fractionation and randomized adapters were obtained from IDT and are listed in Supplementary Table S2.