Truseq stranded total rna lt kit with ribo zero gold

Total RNA samples extracted from peripheral blood of three patients and three unrelated male controls were used to build cDNA libraries using the TruSeq^®Stranded Total RNA LT- kit (with Ribo-Zero TM Gold) (Illumina, USA). Sequencing was performed on the NextSeq 500 platform Mid Output v2 Kit (150 cycles) (Illumina, USA). The FASTQ files were aligned against the ribosomal reference sequence (NCBI, 12/2017) using the BWA software [26] version 0.7.17-r1188, in MEM mode, with the standard parameters, except for the -t 4 parameters. Reads not aligned to ribosomal sequences went to the alignment step against the reference sequence of the human genome (version GRCh37 - hg19) using the STAR software [27], version 2.6.1a_08–27. The annotation database (GTF file) used was the Ensembl file in version 87 in the same build as the human genome reference (GRCh37).

RNA libraries were generated through TruSeq Stranded Total RNA LT Kit with Ribo-Zero TM Gold (RS-122-2301) for Illumina sequencing as per the manufacturer’s protocol. The total RNA was extracted and subjected to the Ribo-Zero rRNA Kit for the removal of rRNA (ribosomal RNA). Linear RNAs were removed with RNase R (Epicenter, Madison, WI, United States); to break down circRNAs into fragments. Random hexanucleotide primers were utilized to create first-strand cDNAs using the circRNA fragments as templates. Then, second-strand cDNAs were generated using a second-strand cDNA synthesis mix following the dUTP mix as a substitute for dTTP. At this point, different adapters were ligated at 5′ and 3′ ends of the second strand. The cDNA strand containing dUTP was digested using the uracil N-glycosylase (UNG) enzyme, preserving only the cDNA strand with various adapters. The recovered cDNA was further purified utilizing a cDNA purification kit. After purification, a poly adenylation poly (A) tail was added to cDNA and a sequencing adaptor was ligated at the repair end. Afterward, size selection and PCR amplification were performed. The Agilent 2100 Bioanalyzer was used to examine the quality of the prepared library. After ensuring the quality, Illumina HiSeq^TM 2500 was used for library sequencing.