stress_3
A total of 1 ?g of RNA per tumor sample from the NS and S groups were used as input material for RNA sample preparation. Sequencing libraries were generated using the NEBNext Ultra RNA Library Prep Kit from Illumina, following the manufacturers recommendations. To preferentially select cDNA fragments of 250-300 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 ?L of USER enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37C for 15 min followed by 5 min at 95C before PCR. The PCR was performed with 0.2 l Phusion High-Fidelity DNA polymerase (Catalog # M0530S, NEB), universal oligo-dt primer [d(T)23VN]. Lastly, the products were purified with the AMPure XP system, and library quality was assessed on the Agilent Bioanalyzer 2100 system.