Bulk RNA-seq
The concentration and integrity of total RNA were evaluated using a Qubit RNA HS Assay Kit (Thermo Fisher Scientific, catalog no. Q32852) and Agilent RNA 6000 pico kit (Agilent, catalog no. 5067-1513). RNA sample libraries were prepared using an MGI ribosomal RNA removal kit (MGI, catalog no. 1000005953) and MGIEasy RNA Library Prep Set (MGI, catalog no. 1000006383) in accordance with the manufacturer’s manual. The concentrations of the libraries were quantified using a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, catalog no. Q32851) and the quality of the libraries was evaluated using the Agilent DNA 1000 Kit (Agilent, catalog no. 5067-1504). Libraries were sequenced on a MGISEQ-2000 sequencer (MGI) with 100 bp paired-end reads. First, adapters and low-quality sequences were filtered, then qualified reads were aligned to the GRCh38 reference genome using STAR v.2.5.1b. Picard Tools (v.1.84) was used to remove duplicated reads.