Nanodrop nd 2000 ultra micro spectrophotometer

Total RNA from cultures of FH215-D2B6, FH215-trc, FH215-F19 and FH215-J23119 was isolated using the RNAprep Pure Cell/Bacteria Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. RNA quality and quantity were analyzed using a NanoDrop ND2000 ultramicro spectrophotometer (Thermo Scientific, USA). The cDNA was synthesized from the total RNA using the All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TransGen, Beijing, China), and qPCR was performed with NovoStart^® SYBR qPCR SuperMix Plus (Novoprotein, Beijing, China) according to the manufacturer’s instructions. A negative control without added cDNA was used to check the purity of samples for contaminating genomic DNA. All qPCR assays were performed in triplicate on a 7500 Fast Real-Time PCR System (Applied Biosystems, USA). The cycling parameters were as follows: denaturation step at 95 °C for 1 min followed by 40 cycles of 95 °C for 20 s and 60 °C for 1 min.

Total RNA was extracted from the abdominal fat tissue samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA concentration and quality were measured at OD 260/280 using the Nanodrop ND-2000 ultra-micro spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). OD 260/280 in the range of 2.0–2.2 and RNA concentration around 500 ng/μL. The integrity of the RNA was measured by analyzing 2 µL of the total RNA on a 1% agarose gel. RNA libraries were then constructed using the TruSeq RNA-Seq Library Prep Kit v.2 (Illumina, Shanghai, China) following the manufacturer’s instructions. Subsequently, libraries were constructed and sequenced using a HiSeqTM 2000 instrument (Illumina), completed by Gene Denovo Biotechnology Co., Ltd. (Guangzhou, China).
To ensure high-quality clean reads, FASTP was used for quality control. The resulting high-quality clean reads were compared with a ribosomal database using Bowtie2 [15 (link)]. This comparison allowed for calculating the ratio of high-quality clean data to total RNA. The reads derived from the filtered ribosomes were aligned to the reference genome using TopHat2 [16 (link)]. Unmapped reads were extracted from the alignment results, and both ends of each unmapped read (default 20 bp) were intercepted to obtain the anchor reads. The anchor reads were then aligned to the reference genome.