Qubit 3.0 fluorescence quantifier

In accordance with the instructions of fast DNA library preparation kit, the hotspot mutation panels of 50 tumor-associated genes designed for specific genomic regions or targets were added initially based on the multiplex polymerase chain reaction (PCR) library construction technology, and then multiplex PCR enrichment was performed on specific gene region fragments. Next, the obtained target gene fragments were subjected to end repair, barcode adapter ligation, and PCR amplification (Eastwin Scientific Equipment, Suzhou) to prepare a library. Afterwards, the library was quantified with Qubit 3.0 fluorescence quantifier (Thermo Fisher Scientific, Shanghai), and the fragment size of the library was analyzed with the Bioptic Qsep 100 automatic nucleic acid analyzer (Bioptic Inc., Jiangsu) to evaluate the library quality. Finally, a high-throughput sequencer (MGI, Shenzhen) was used for sequencing, and the data splitting, denoising, and comparison were accomplished in the Linux system to obtain the gene mutation results of various samples.
The library construction and NGS were carried out in Cowin Bio., Jiangsu.

The 3 μg of RNA per sample was used as the input material for RNA sample preparation. mRNA was separated from total RNA and purified by mRNA Capture Beads (Vazyme, Nanjing, China). The purified mRNA was incubated in a preheated Polymerase chain reaction (PCR) instrument (Life Technologies, CA, USA) at 94°C for 7 min, interrupted, cooled immediately after the interruption, and centrifuged. The first strand of cDNA was synthesized using the supernatant containing fragment RNA as the template. The double‐strand synthesis reaction reagents were added to the first strand product of the synthesized cDNA, mixed, centrifuged, and incubated in a metal bath to synthesize the second strand. Magnetic beads were used to purify the double‐strand cDNA product. After adapter ligation, the fragments were screened by DNA clean beads (Vazyme, Nanjing, China), and the library was enriched by PCR using the screened cDNA fragments as templates.
Qubit 3.0 fluorescence quantifier (Thermo Fisher Scientific, Waltham, MA, USA) was used for preliminary quantification until the concentration reached more than 1 ng/μL. The Qsep400 high throughput analysis system was used to detect the inserted fragments of the library. After the pieces were inserted as expected, Q‐PCR was used to accurately quantify the effective concentration of the library (effective concentration>2 nM).