Hiseq 3000 sequencing platform

MHCC-97H cells transfected with NUPR1 overexpression vector or control vector were seeded in 6-well plates and exposed to 8 Gy irradiation. After 24 h, total RNA was isolated and subjected to the construction of RNA-seq libraries. The quality of the RNA libraries was evaluated using the Agilent 2200 TapeStation (Agilent Technologies, USA). Library sequencing was performed on a HiSeq 3000 sequencing platform (Illumina Company, USA) by Guangzhou RiboBio Corp., China.

Sugarcane leaf blight-susceptible sugarcane cultivar Yuetang93-159 was planted in Fuzhou, China (26°5′0″ N, 119°13′45″ E) and the S. tainanensis strain StFZ01 was collected from its leaves with the typical symptoms of SLB. After the isolates were testified using ITS detection (Figure 1) and by pathogenic morphology [24 ], one assigned as StFZ01 was used for genome sequencing and analysis. The fresh mycelia cultivated on potato dextrose broth (PDB) media was collected for DNA and RNA extraction. For long-read genomic sequencing, high-quality genomic DNA was extracted using Ligation Sequencing Kit (SQK-LSK110), then BluePippin DNA size selection system was used to select large DNA fragments (>20 kb) for sequence library preparation following the manufacturer’s instructions, and the sequencing was conducted on PromethION sequencing platform from Oxford Nanopore Technologies (ONT). For Illumina short-read sequencing, genomic DNA and mRNA were extracted, purified, and prepared for sequencing libraries using Illumina DNA Prep Kits (Illumina, Inc., San Diego, CA, USA) and Illumina Stranded mRNA Prep (Illumina, Inc., San Diego, CA, USA), respectively. Illumina genomic DNA sequencing and RNA-seq were performed on the Illumina HiSeq 3000 sequencing platform (350 bp library and PE150 strategy).

Total RNA from control and PELP1-KD MDA-MB-231 cells was isolated using the RNeasy mini kit (Qiagen, Hilden, Germany). RNA-seq was performed using the Genome Sequencing Core (UT Health SA) established protocol and sequencing was done using 50 bp single read sequencing module with Illumina HiSeq 3000 sequencing platform [25 (link)]. Differential gene expression analysis was done in DEseq2 [26 (link)] and genes with fold change >2 and adjusted p-value < 0.05 were categorized as differentially expressed genes. RNA-seq data was deposited in the GEO database with a GEO accession number (GSE191066).

To screen out candidate genes that mediate the food-driven resetting of the liver clock, the mice were subjected to fasting/re-feeding cycles or DD for 6 weeks as described above. Liver samples were pooled, and total RNA was isolated for the construction of RNA-seq libraries. The quality of the RNA libraries was evaluated using the Agilent 2200 TapeStation (Agilent Technologies, USA). Library sequencing was performed on a HiSeq 3000 sequencing platform (Illumina Company, USA) by Guangzhou RiboBio Corp., China. Fragments Per Kilobase of transcript per Million mapped reads (FPKM) were calculated for additional statistics. All the reads were mapped to the mouse genome (GRCm38/mm10).

Liver samples were collected to screen out candidate genes that mediate the role of POLB in regulating the liver clock and HCC. Total RNA was isolated from the liver samples for construction of RNA-seq libraries. The RNA libraries were sequenced using a HiSeq 3000 sequencing platform (Illumina Company, USA) by RiboBio (Guangzhou, Guangdong, China). For additional statistics, Fragments Per Kilobase of transcript per Million mapped reads (FPKM) were calculated. All reads were mapped to the mouse genome (GRCm38/mm10). The DAVID tool was used for pathway enrichment and gene cluster analysis (https://david.ncifcrf.gov/).