Nanodrop 8000 uv vis spectrometer

Total RNA was isolated from fresh frozen tissues of mouse stomach sacrificed 3 days after tamoxifen or vehicle administration (n=3/group) using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Total RNA quality and quantity were determined using a Nanodrop 8000 UV-Vis spectrometer (Thermo Fisher Scientific) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The quantity of all RNA samples was >2 μg, and the quality had an RNA integrity number value >8.0.
Library construction for whole transcriptome sequencing was performed using the TruSeq RNA sample preparation v2 kit (Illumina, San Diego, CA, USA). Isolated total RNA (2 μg) was used for reverse transcription reactions with poly (dT) primers and SuperScript^TM II reverse transcriptase (Invitrogen/Life Technologies, Grand Island, NY, USA) according to the manufacturer’s protocols. Briefly, the RNA sequencing (RNA-seq) library was prepared through cDNA amplification, end-repair, 3’ end adenylation, adapter ligation, and amplification. The quality and concentration of the library were measured using Bioanalyzer and Qubit instruments, respectively. Sequencing of the transcriptome library was carried out using the 100 bp paired-end mode of the TruSeq Rapid PE Cluster kit and the TruSeq Rapid SBS kit (Illumina).

Formalin-fixed paraffin-embedded (FFPE) sections were deparaffinized and lysed with proteinase K for 4 h, and genomic DNA was extracted using a Maxwell 16 CSC DNA FFPE kit and Maxwell MDx automation instrument (Promega Corporation, Fitchburg, WI, USA). DNA concentration and purity of all genomic DNA samples were measured using a Nanodrop 8000 UV-Vis spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) and Qubit 2.0 Fluorometer (Thermo Fisher Scientific). To estimate DNA degradation, DNA median size and ΔCt value were measured by 2200 TapeStation Instrument and real-time polymerase chain reaction (PCR) (Agilent Technologies, Santa Clara, CA, USA). To improve sequencing quality, we used strict sample quality thresholds for extracted DNA: 1) purity: 260/280>1.8, 260/230>1.8; 2) total amount >250 ng; and 3) degradation: ΔCt value <2.0 or DNA median size >0.35 kb.