Bulk RNA-Seq of TREM1 deficient LCSLCs of Huh7 and Hepg2 cell lines
Bulk RNA-Seq of TREM1 deficient LCSLCs of Huh7 and Hepg2 cell lines
Cells were harvested using trypsin-EDTA to ensure a single-cell suspension. Cells were washed by centrifugation at 1500 rpm for 5 minutes at 4°C and resuspended in ice-cold FACS buffer. Cell viability was confirmed to be above 90%. Fc block was performed by adding 100 µL of Fc block diluted in FACS buffer (1:50 ratio) to each sample, followed by incubation on ice for 20 minutes. Cells were stained with FITC anti-CD133 and APC anti-EpCAM antibodies diluted in FACS buffer at 4°C for 45 minutes in dark. Cells were washed three times by centrifugation at 1500 rpm for 5 minutes and resuspension in ice-cold FACS buffer. Cells were sorted using Invitrogen™ Bigfoot™ Cell Sorter (Thermo Fisher Scientific). Cells were sorted based on CD133 and EpCAM expression. Sorted cells were collected into appropriate collection tubes, and the purity of sorted populations was analyzed, aiming for at least 90% purity. Unstained, single-stained, and isotype controls were included for accurate gating. Total RNA was isolated using RNeasy Mini kit (74104, Qiagen).  Briefly RNA sequencing was carried out using an Illumina NovaSeq 6000, and filtered clean reads were mapped to a reference genome using hierarchical indexing for spliced alignment of transcripts 2 (HISAT2). Visualization of alignment results were verified using Integrative Genomics Viewer.  Differential expression analysis was performed using DESeq with Fold Change ≥1 and FDR < 0.05 set as screening criteria