Ribo off rrna depletion kit human mouse rat

We prepared the sequencing libraries following the manufacturer’s recommendations of Ribo-off rRNA Depletion Kit (Human/Mouse/Rat) (Vazyme Biotech Co., Ltd., Nanjing, China, N406) and VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina (Vazyme Biotech Co., Ltd., Nanjing, China, NR605). The details of library construction are as follows. First, we removed ribosome RNA from 200 ng total RNA using Ribo-off rRNA Depletion Kit (Human/Mouse/Rat) (Vazyme, N406). Then, we fragmented the RNA into small pieces using divalent cations at elevated temperatures. The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random primers, followed by second-strand cDNA synthesis using DNA Polymerase I, RNase H, deoxyuridine triphosphate (dUTP), deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP) and deoxycytidine triphosphate (dCTP). Then, a single ‘A’ base and the adapters were subsequently added to these cDNA fragments. In order to select the appropriate cDNA fragment size for sequencing, we selected the library fragments with VAHTSTM DNA Clean Beads (Vazyme, N411). The polymerase chain reaction (PCR) amplification was performed, and the aimed products were finally purified. After cluster generation, the libraries were sequenced on an Illumina novaseq 6000 platform, and the raw fastq files of 150-bp paired-end reads were generated.