Neb kits

Total RNA was extracted from samples from 10 OS patients and 10 healthy controls using phenol-chloroform (TRIzol; Invitrogen; ThermoFisher Scientific, Inc., Waltham, MA, United States). The quality of RNA was assessed by capillary electrophoresis (Agilent Technologies, Inc., Santa Clara, CA, United States). Libraries for small RNA sequencing were prepared using NEB kits (New England Biolabs, Inc., Ipswich, MA, United States). qRT-PCR with SYBR-Green (Takara, Osaka, Japan) to detect CDC5L, CUL1, CXCL10, EIF2AK2, POLR2B, PTEN, STAT1, and TBP expression levels, GAPDH was applied as a house keeping gene. The reaction was performed via 40 amplification cycles using the following protocol: 95°C for 3 min, 95°C for 45 s, 55°C for 15 s, and 72°C for 50 s. Primers used in PCR were shown in Supplementary Table S1. Samples were analyzed in triplicate, and gene expression was quantified by normalizing target gene expression to that of the internal control using the 2^−ΔΔCt formula.

MinION library construction used the 1D sequencing kit (SQK-LSK108-Oxford Nanopore; Oxford, UK). Two 12-barcoded amplicons (1,800 ng total in each) were combined, end-repaired, and dA-tailed in accordance with ONT instructions using NEB kits (New England Biolabs, Ipswich, MA, USA) and the modified Ampure bead purification described above. Ligation of the ONT adaptor used the Blunt/TA ligase master mix (NEB) with an addition of 1 µL of freshly prepared ATP solution (∼4 mg/mL) to facilitate ligation. All libraries were analyzed on R9.4 flow cells. To determine the contribution of rRNA operon sequences from PCR reagents and ONT sequencing kits, it was necessary to sequence the PCR negatives from our amplifications. Unfortunately, the original PCR negative results were accidently discarded and the LSK 108 kit that we used for these particular studies is no longer commercially available. Therefore, to address this “kitome” issue, we returned to the original DNA from Subject 15 (BAL, throat, mouth, and nose), re-amplified as described in the Methods section, and performed a sequencing reaction on both PCR-negative and PCR-positive samples with the LSK 109 kit. Analysis of this “kitome” indicated a 4,000-fold difference in sequence read numbers passing QA/QC (13 negative reads vs 41,135 positive reads), representing a possible contamination of 0.03% (Supplementary Fig. S5).

MinION library construction employed the 2D sequencing kit. Twelve barcoded amplicons (1200 ng total) were combined, end-repaired, dA-tailed as per ONT instructions using NEB kits (New England Biolabs, Ipswich, MA, USA) and the modified AMPure bead purification described above. Ligation of the ONT adaptor and hairpin employed the Blunt/TA ligase master mix (NEB) with an addition of 1 µl of freshly-prepared ATP solution (~4 mg/ml) to facilitate ligation. All libraries were analyzed on R9 flow cells.