Con335

Lentivirus of negative control (CON077) and LV-PRNP-RNAi (hU6-MCS-Ubiquitin-EGFP-IRES-puromycin), negative control (CON335) and LV-PRNP (Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin) were constructed by GENECHEM Biotech Co., Ltd. The HESC were digested and uniformly plant in 6-well plate, when the cells were cultured for 24 h, attached to the bottom of flask and grew to confluence of 50%, lentivirus and infection enhance reagent were added into the culture medium, and we exchanged the medium after 12 h of infection. 72 h later, the cells were inspected under the fluorescence microscope and analyzed by qRT-PCR or western blotting assay for evaluating the infection efficiency, then the infected cells were treated by puromycin for 7 days to select out the successfully infected cells.

Lentivirus of negative control (CON335) and LV- LGALS9 (30842-14) were constructed by GeneChem Corporation (Shanghai, China). Transfection was performed according to the manufacturer’s protocol. Briefly, THP-1 monocytic cells were incubated in 1640 medium with 10% FBS. The cells were then transduced with the recombinant lentivirus in the presence of polybrene. Stably transfected cell lines expressing GFP were screened using puromycin. LGALS9 expression was subsequently evaluated using real-time PCR. The primers used for human LGALS9 were sense, 5′-TCTCCAGGACGGACTTCAGA-3′ and anti-sense, 5′-CACCAGGAAGCAGAGGTCAA-3′. The primers for the internal reference gene ACTB were sense, 5′-GCGTGACATTAAGGAGAAGC-3′ and anti-sense, 5′-CCACGTCACACTTCATGATGG-3′. Total RNA of cell lines from the control group THP-1_NC and experimental group LGALS9_OE were extracted using a RNeasy Mini Kit (Qiagen). Three replicates were used per treatment group in this study. After cDNA library preparation, the libraries were sequenced on Illumina HiSeq 2500 and the resulting FASTQ files were aligned to the human genome (GRCh38.74). Gene expression profile for the individual samples was thereafter calculated as RPKM values.