El transfection reagent

PC3 cells were plated at a 6-well plate with 3.5 × 10⁵ cells per well. After overnight culture, 250- pmol siNC or siASH1L#1 were transfected with 10 μl of the EL transfection reagent (TransGen Biotech, catalog no. FT201) per well as dictated by the protocol. The cells were collected at 48 h; 10% of the cells were treated with the RNAiso Plus reagent, and the remaining cells were quickly frozen with liquid nitrogen. The knockdown efficiency of the ASH1L gene was confirmed by qRT-PCR after mRNA extraction. The frozen samples were mailed to BGI Bioinformatics Corporation (Wuhan, China) with the project number: F21FTSNCKF3033_HONgbclN. The RNA preparation, library construction, and sequencing using the DNBSEQ platform were performed at BGI. RNA sample quality was examined by Bioanalyzer (Agilent 2100). After getting clean reads, Bowtie2 (version: v2.2.5) was used to map the clean reads to the reference gene sequence (transcriptome), and the gene expression level of each sample was calculated with RSEM (v1.2.8). The DEseq2 method was used for differential gene detection, and according to results of the differential gene detection, the R Package pheatmap was used to perform hierarchical analysis on the union set differential genes. The data were analyzed using the BGI Dr. Tom multi-omics integration analysis website (https://biosys.bgi.com/#/report/login).

siRNAs (double-stranded RNA oligonucleotides) (Genepharma, China) were used to knockdown the expressions of RNF11 and ZMYND11 in DF-1 cells and the sequences were shown in Table 1. siRNA duplexes were transfected into DF-1 cells at a final concentration of 50 nM using EL Transfection Reagent (Transgen Biotech, China). The interference efficiency was detected by qRT-PCR and Western blot as described below.

DF-1 cells cultured on 12-well plate were co-transfected with indicated plasmids (250 ng) and miRNA oligonucleotides (100 nM) using EL Transfection Reagent (Transgen Biotech, China). After 24 h, luciferase activities were determined using the Dual-GLO® Luciferase Assay System Kit (Promega, USA) following the protocol supplied. The relative luciferase activities were calculated by dividing Renilla luciferase values by internal control firefly luciferase values.