ABSTRACT: Purpose: to study the transcriptional changes that are induced by activating NR2F1 using an agonist (C26) that we developed. Methods: T-HEp3-GFP cells were pretreated with DMSO or 0.5 µM C26 for 6 days then inoculated on CAM and treated every 24 hours. After 7 days, tumors were collected and dissociated into single cells. FACS was used to isolate T-Hep3 tumor cells using GFP. RNA was extracted from fresh frozen cell pellets using Qiagen RNeasy Plus Universal mini kit following manufacturer’s instructions (Qiagen, Hilden, Germany). RNA samples received were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina following manufacturer’s instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented for 15 minutes at 94°C. First strand and second strand cDNAs were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. The sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were clustered on 1 lane of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq instrument (4000 or equivalent) according to manufacturer’s instructions. The samples were sequenced using a 2x150bp Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification. Three replicate samples per group were used . R (v.4.0.3) was used to perform bioinformatics analysis. Quality control was performed using FastQC (v0.11.8). Trim Galore! (version 0.6.5) was used to trim the adapter sequences with a quality threshold of 20. The human genome reference used was GRCh38.p13 and GENCODE release 36. Alignment was performed using STAR aligner (v2.7.5b). Gene level read counts were obtained by using Salmon (v1.2.1) for all libraries . All samples passed the quality control requirements with > 60% of reads uniquely mapping (>20M uniquely mapped reads for each library) using STAR. Differential expression analysis was performed using the gene level read counts and the DESeq2 (v1.28.1) R package. Heatmaps show the z-scores of gene-level read counts and were generated using heatmaply (v1.1.0). Genes with adjusted p-value less than 0.05 were considered differentially expressed.
