title: GSM7783217: D80, 2, CMZ, bulk RNA; Homo sapiens; RNA-Seq
library_construction_protocol: Wash the samples twice with PBS, add 0.5 ml of digestion solution (containing 0.25% Trypsin-EDTA, 50 μg/ml DNase I, 20 μg/ml Y-27632), and digest at 37°C for 10 minutes. Add 1 ml of culture medium to neutralize the digestion, filter through a mesh filter, centrifuge at 1000 rpm for 5 minutes, remove the supernatant, and resuspend in an appropriate volume of suspension solution (containing PBS, 50 μg/ml DNase I, 20 μg/ml Y-27632). Count and calculate the proportion of viable cells (≥90%), adjust the cell concentration to 1000 cells/μL, and keep on ice for further testing For each sample, total RNA was extracted using TRIzol reagent and further purified using the RNeasy Mini Kit (Qiagen), following the manufacturer's instructions. The quality and quantity of the RNA were evaluated using a NanoDrop 2000 spectrophotometer, Agilent 2100 Bioanalyzer, and Agilent RNA 6000 Nano Kit. Annoroad Gene Technology conducted the RNA library construction and RNA sequencing (RNA Seq). To generate the sequencing libraries, the NEB Next Ultra RNA Library Prep Kit for Illumina (NEB) was used, and library clustering was performed with the HiSeq PE Cluster Kit v4-cBot-HS (Illumina) according to the manufacturer's recommendations. Following cluster generation, the libraries were sequenced on an Illumina platform, generating 150 bp paired-end reads.