Small rna kit

The adapter was designed (5’ to 3’) with a 4 nt fork, a T7 promoter, the 5’ Illumina adapter (as used in the Illumina small RNA kit), a 3 nt UMI (unique molecular identifier), an 8 nt unique barcode followed by CA. The Dam-RING1B mESCs were processed with different adapters. These contained a 6 nt fork, a 6 nt unique barcode followed by GA. The barcodes were designed with a hamming distance of at least two between them. Bottom sequences contained a phosphorylation site at the 5’ end. Adapters were produced as standard desalted oligos. Top and bottom sequences were annealed at a 1:1 volume ratio in annealing buffer (10 mM Tris pH 7.5–8.0, 50 mM NaCl, 1 mM EDTA) by immersing tubes in boiling water, then allowing to cool to room temperature. The oligo sequences can be found in Supplementary Table 2.

Wild type Komagataella phaffii strain NRRL Y-11430 was cultivated in 5 mL of rich defined medium (RDM)^{45 (link)} with 4% glycerol by volume. Five identical cultures independently cultivated were used as biological replicates. Cultures were inoculated at OD600 = 0.1, and grown at 30°C for 24 hours until OD600 ≈ 16. Total RNA was extracted using the Qiagen miRNA extraction kit. RNA was converted to cDNA libraries using the New England Biolabs Small RNA Kit and sequenced on an Illumina NextSeq. Reads were aligned to the K. phaffii genome⁴⁶ using Salmon.^{47 (link)} Small RNAs were defined as continuous regions of >50 read depth. Expression of each small RNA was calculated using transcripts per million (tpm), normalized by sample.
To quantify edge resolution of each small RNA, we defined each edge of the RNA as the 20 bp upstream and downstream of the annotated boundary. Over this region, we calculated the change in read depth at each base pair. We defined edge resolution as the ratio of the maximum change in read depth to the maximum read depth.
$$\mathit{\text{Edge}}\mathit{\text{resolution}}=\frac{\text{max}\left(\Delta \mathit{\text{read}}\mathit{\text{depth}}\right)}{\text{max}\left(\mathit{\text{read}}\mathit{\text{depth}}\right)}$$
The edge resolution score was then calculated as the mean of the 5’ and 3’ edge resolution for each small RNA. An edge resolution of 1 indicates that all paired end reads start and end at the same 5’ and 3’ base pairs.

Small RNA was isolated using miRNeasy Mini Kit (Qiagen, Germantown, MD) and sequenced following the Small RNA Sample Preparation Protocol (Illumina, San Diego, USA). The library was prepared from 10 ng of total RNA per sample according to the manufacturers instructions (TruSeq, Small RNAKit, Illumina). Single-stranded cDNAs were created with SuperScriptII Reverse Transcriptase and double-stranded cDNAs generated by PCR using adapter specific primers. Purified libraries were quantified and qualified using the High Sensitivity DNA Kit on a 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). Sequencing of the libraries was carried out at the Genomic Core Facility (Cleveland Clinic) utilizing HiSeq2000 (Illumina). After sequencing, the data were obtained in Illumina FASTQ format (Illumina). The data were analyzed using NGS small RNA sequence analysis software (version 2.5.1) using the rat build rn4MiRNA program (see Supplemental Figure 1 for description of data analysis pipeline). NGS data have been deposited in NCBI’s Gene Expression Omnibus and are awaiting assignment of their accession number.