Kapa rna hyperprep kit with riboerase hmr

Immediately following each brush mucosal cell collection, the brush was immersed in Qiazol (cell lysis Reagent from Qiagen, USA), mixed well, and stored temporarily in a –80° C freezer at CTRC-CRC. The next day, all the completed brush mucosal samples were delivered on dry ice to Roswell Park’s DataBank and BioRepository (DBBR) Shared Resource for storage at –80° C. After sample collection was completed from all participants, they were delivered on dry ice to the Roswell Park Genomics Shared Resource for RNA extraction and RNA-seq assay. Purified RNA was prepared using the miRNeasy micro kit (Qiagen, Cat No. 217084) and sequencing libraries were prepared with the KAPA RNA HyperPrep Kit with RiboErase (HMR) (Roche, USA), from 100ng total RNA. The final RNA-seq libraries were sequenced on Illumina NovaSeq 6000 using 100 paired end sequencing.

RNA-seq libraries were prepared using 800 ng of total RNA by KAPA RNA HyperPrep RiboErase and mRNA kits. Ribosomal RNA was depleted, poly(A) mRNA was selected by Sera-Mag™ Magnetic Oligo(dt) particles (GE38152103011150) respectively, and then fragmented to around 200 to 300 bp (base pairs) with heat in fragmentation buffer. cDNA synthesis, end-repair, A-tailing, and ligation of the New England Biolabs (NEB) adapters were all performed following the KAPA RNA Hyper protocols (KAPA RNA HyperPrep Kit with RiboErase (HMR) and KAPA mRNA HyperPrep kit, Roche). Libraries were size-selected for 300 to 500 bp fragments by double AMPure beads- and PCR-amplified using 2× KAPA HiFi HotStart mix and NEB dual indexes. Library quality (concentration and product size) was measured on an Agilent 2100 Bioanalyzer (DNA 1000 chip). Paired-end libraries were sequenced with the Illumina NovaSeq 6000, (paired-end 2 × 151 nucleotide read length) with sequence coverage of 30 to 40 million paired reads. and data analysis was performed as previously described (16 (link)).

Only samples from collection and handling methods passing the prespecified quality metrics (average DV₂₀₀ > 30%) and with individual RNA content higher than 50 ng were submitted to library preparation and sequencing.
Libraries were prepared using KAPA RNA HyperPrep Kit with RiboErase (HMR), (Roche, Basilea, CH) using the manufacturers protocol with an input of 50–100 ng RNA. All libraries were quality assessed for fragment size (Bioanalyser) and quantified using the NEBNext Library Quant kit (New England Biosciences, Herts, United Kingdom). Normalized libraries were loaded onto a NovaSeq SP flow cell (Illumina, SanDiego, US) and sequenced to a total read length of 2 × 76 bp. The quality of the sequencing was confirmed for number of clusters passing filter, Q30 scores, error rate, cluster density and read distribution. Raw sequence data in FASTQ format were input to QC assessment steps hosted on the DNAnexus cloud platform. Basic sequencing metrics such as GC content were calculated from unaligned reads using FastQC (Leggett et al., 2013 (link)). Read alignment to the human reference genome GRCh38 was then performed using StarAlign (Dobin et al., 2013 (link)), with outputs used to calculate post-alignment QC metrics such as duplication rate [calculated using Picard MarkDuplicates (http://broadinstitute.github.io/picard/)] and coverage of the housekeeping genes.

Cryopreserved PBMC samples from Children’s Oncology Group (COG) B-ALL study AALL-1331 were obtained from the COG Biobank. All subjects provided consent for banking and future research use of these specimens in accordance with the regulations of the institutional review boards of all participating institutions. Samples were resuspended in TRIzol reagent (Life Technologies) then processed using 5prime Phase Lock Gel Heavy tubes (Quantabio) following the manufacturer’s instructions. Following precipitation, the RNA was quantitated using the Qubit RNA BR assay kit (Life Technologies) and evaluated for quality using the Agilent 2100 Bioanalyzer with the RNA nano chip (Agilent). The KAPA RNA HyperPrep Kit with RiboErase (HMR) (Roche) with 100–200ng of total RNA was used for library construction with either Nextflex-96 RNA barcode adapters (BIOO Scientific, Illumina HiSeq 4000 platform), or IDT for Illumina unique dual indices adaptors (Illumina, NovaSeq 6000 platform) and 12 PCR cycles of library amplification. Libraries were evaluated on the Bioanalyzer 2100 using the DNA 1000 chip. Libraries were sequenced using either the HiSeq 4000 (Illumina) or NovaSeq 6000 (Illumina) paired-end 2X150 cycle protocol.