Kapa rna hyperprep kit with riboerase

For paRNA-seq, we purified poly(A) RNA from 1 µg of ES total RNA using a NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB). For rdRNA-seq, we depleted rRNA from 1 µg of ES total RNA using a GeneRead rRNA Depletion Kit (Qiagen). We prepared conventional RNA-seq library DNA with the resulting RNA using a commercial kit (NEBNext Ultra Directional RNA Library Prep Kit for Illumina; NEB) in accordance with the manufacturer’s protocol, with slight modifications during RT and PCR steps. We used SuperScript III (Thermo Fisher) for RT. We used KAPA HiFi DNA polymerase (Kapa Biosystems) in the PCR step. When we performed rdRNA-seq using 10 or 1 ng of ES total RNA, we utilized a KAPA RNA HyperPrep Kit with RiboErase (Kapa Biosystems) for the preparation of sequencing library DNA according to the manual instructions.

RNA was isolated from peripheral blood buffy coat samples on the automated Chemagic 360 (Perkin Elmer) instrument according to the manufacturer’s instructions. Extracted RNA was quantitated by Qubit^™ RNA BR Assay Kit (Thermo Fisher Scientific) followed by the RNA quality check using Fragment Analyzer (Agilent). For each sample, RNA libraries were prepared from 100ng RNA using the KAPA RNA HyperPrep Kit with RiboErase (Kapa Biosystems) according to manufacturer’s protocol, followed by quality check using Fragment Analyzer (Agilent) and quantification by qPCR (Kapa qPCR quantification kit, Kapa biosystem) on the LightCycler 480 (Roche). The libraries were normalized and pooled, and then sequenced using NovaSeq6000 platform (Illumina) to an average of 40M 101bp paired end reads per sample. Low-quality reads and adapter sequences were trimmed from the raw sequencing data with Trimmomatic [32 (link)]. The remaining reads were then aligned to human reference genome hg38 with STAR aligner [33 (link)]. Gene counts were quantified with the STAR --quantMode GeneCounts function. Fragments per kilobase of transcript per million mapped fragments (FPKM) were quantified with Cufflinks [34 (link)].

For RNA isolation 1∗10⁶ isolated monocytes were resuspended in 350 μL of RNA later Buffer (QIAGEN). RNA was isolated using RNeasy kit (QIAGEN) including DNaseI (QIAGEN) digestions.
Total RNA isolated from monocytes was used for the preparation of the RNA sequencing libraries using the KAPA RNA HyperPrep Kit with RiboErase (KAPA Biosystems). In short, oligo 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. First strand synthesis, second strand synthesis and A-tailing was performed according to protocol. For the adaptor ligation, a 1.5 μM stock was used (NextFlex DNA barcodes, Bioo Scientific). First and second post-ligation cleanup was performed according protocol. A total of 11 PCR cycles were performed for library amplification. The library amplification cleanup was done using a 0.8x followed by a 1.0x bead-based cleanup. Library size was determined using the High Sensitivity DNA bioanalyzer kit, and the library concentration was measured using the dsDNA High Sensitivity Assay (Denovix). Paired-end sequencing reads of 50 bp were generated using an Illumina NextSeq 500.

For RIP-Seq samples, 9 μl of RIP sample (from 15 μl total) were used. Each library preparation included 1 μl of 1:250 dilution of ERCC Spike-In RNAs (Ambion, #4456653). 10 μl total were prepped using the KAPA RNA HyperPrep Kit with RiboErase (Kapa Biosystems; product #KR1351). Sequencing was performed on an Illumina Next-Seq 500, using high-output, 75-cycle kits (Illumina, #20024906).

Bulk RNA sequencing was performed on melanoma tumors, as previously described^{37 (link),38 (link)}. In brief, RNA was extracted from tumors using the RNeasy PowerLyzer Tissue & Cells Kit (QIAGEN) and quality was assessed with a 2100 Bioanalyzer System (Agilent Technologies). Ribosomal RNA was depleted Kapa RNA HyperPrep Kit with RiboErase (Kapa Biosystems, Inc., Cape Town, South Africa). Sequencing libraries were generated using TruSeq RNA sample prep kits (Illumina) and sequenced on an Illumina HiSeq 2500 using 2 × 150 bp paired-end reads with a target of 20–25 million reads per sample. Raw sequencing files were aligned to the GRCh38 human genome.

Total RNA was isolated using Direct-zol RNA Miniprep Plus (Zymo Research, Irvine, CA, USA). RNA sequencing libraries were generated with the Kapa RNA HyperPrep kit with RiboErase (Kapa Biosystems, Wilmington, MA, USA; KR1351), according to the manufacturer’s protocol. Total RNA (250 ng) from each sample was used for sequencing library preparation. The final libraries were validated with the Agilent Bioanalyzer High Sensitivity DNA kit (Agilent Technologies, Santa Clara, CA, USA). Sequencing was performed on the Illumina NovaSeq6000 with S4 Reagent Kit v1.5 (Illumina, San Diego, CA, USA; 20028313) with a sequencing length of 2 × 101. Real-time analysis (RTA) 3.4.4 software was used to process the image analysis.