Kapa rna hyperprep kit with riboerase human mouse rat hmr

PBMCs were lysed with Qiagen RNeasy lysis buffer (RLT) following 24 h of stimulation and stored at −80°C until isolation. RNA was isolated using the Qiagen RNeasy kit, following the manufacturer’s instructions. A total of 200 ng RNA per sample was used for the preparation of RNA sequencing libraries using the KAPA RNA HyperPrep kit with RiboErase (human/mouse/rat [HMR]) (Kapa Biosystems). In short, oligonucleotide hybridization and rRNA depletion, rRNA depletion cleanup, DNase digestion, DNase digestion cleanup, and RNA elution were performed according to protocol. Fragmentation and priming were performed at 94°C for 6 min 30 s. First-strand synthesis, second-strand synthesis, and A-tailing were performed according to protocol. For adapter ligation, a 7-μM stock was used (NEXTflex DNA barcodes; Bioo Scientific). The first and second postligation cleanups were performed according to protocol. For library amplification, 6 cycles were used. Library amplification cleanup was performed using a 0.8× bead-based cleanup. The library size was determined using the high-sensitivity DNA bioanalyzer (Agilent Technologies), the library concentration was measured using the DeNovix double-stranded DNA (dsDNA) high-sensitivity assay. Sequencing was performed using an Illumina NextSeq 500 instrument; 38-bp paired-end reads were generated.

PBMCs were lysed with Qiagen RNeasy lysis buffer (RLT) following 24 h of stimulation and stored at −80°C until isolation. RNA was isolated using the Qiagen RNeasy kit, following the manufacturer’s instructions. A total of 200 ng RNA per sample was used for the preparation of RNA sequencing libraries using the KAPA RNA HyperPrep kit with RiboErase (human/mouse/rat [HMR]) (Kapa Biosystems). In short, oligonucleotide hybridization and rRNA depletion, rRNA depletion cleanup, DNase digestion, DNase digestion cleanup, and RNA elution were performed according to protocol. Fragmentation and priming were performed at 94°C for 6 min 30 s. First-strand synthesis, second-strand synthesis, and A-tailing were performed according to protocol. For adapter ligation, a 7-μM stock was used (NEXTflex DNA barcodes; Bioo Scientific). The first and second postligation cleanups were performed according to protocol. For library amplification, 6 cycles were used. Library amplification cleanup was performed using a 0.8× bead-based cleanup. The library size was determined using the high-sensitivity DNA bioanalyzer (Agilent Technologies), the library concentration was measured using the DeNovix double-stranded DNA (dsDNA) high-sensitivity assay. Sequencing was performed using an Illumina NextSeq 500 instrument; 38-bp paired-end reads were generated.