Komagataella phaffii Small RNA Profiling

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.