Novaseq 6000 sp reagent kit v1

We performed a size selection for 125–163 bp on all libraries using 3% agarose dye-free marker H cassettes on a Pippin Prep (Sage Science, Beverly, MA, USA). Next, the libraries were purified by precipitation using ethanol and resuspended with 10 mM Tris-HCl buffer (pH 8.0) with Tween 20. After dilution, the libraries were quantified using the KAPA Library Quantification Kit (Roche Diagnostics, Diegem, Belgium, KK4854). Healthy donor samples were sequenced using a NextSeq 500 using the NextSeq 500 High Output Kit v2.5 (75 cycles) (Illumina, San Diego, CA, USA). We loaded the library at a concentration of 2.0 pM with 10% PhiX and obtained a total of 268 M reads. We loaded the cancer patient samples on one lane of a NovaSeq 6000 (Illumina, San Diego, CA, USA) instrument at a concentration of 300 pM with 10% PhiX using the NovaSeq 6000 SP Reagent Kit v1.5 (100 cycles) (Illumina, San Diego, CA, USA) (paired-end, 2 × 50 cycles, only the first read was used for subsequent analysis), resulting in 267 M reads. For the chemical modification comparison experiment, we used one lane of a NovaSeq 6000 SP Reagent Kit v1.5 (100 cycles) (Illumina, San Diego, CA, USA, 20028401) (Illumina, San Diego, CA, USA) (1 × 100 bp), loading 300 pM with 10% PhiX, resulting in a total of 548 M reads.

Sequencing spike-ins (Sequins) as internal DNA reference standards (2%, wt/wt) were added to the sample DNA (71 (link)). Fragment libraries were prepared with NEBNext Ultra II DNA Library Prep kit for Illumina (E7645) and NEBNext unique dual index primer pairs (E64405) and a total of eight PCR cycles. The Illumina NovaSeq platform was used for sequencing (NovaSeq 6000 SP reagent kit v1.5, 300 cycles, paired-end reads; Illumina; 20028400).

For RNA-seq, HBVP were treated as indicated. Total RNA was prepared using the Nucleo Spin RNA Mini Isolation Kit with DNA Removal Column (Machery-Nagel, Düren, Germany). RNA-seq was performed at the c.ATG facility of the NGS Competence Center Tübingen (NCCT) using the QuantSeq 3′ mRNA-Seq Library Prep FWD and the NovaSeq 6000 SP Reagent Kit v1.5 with 200 cycles and 15 million clusters (both kits from Illumina, Berlin, Germany). Primary analyses were performed by sequencing data demultiplexing, and secondary analyses using the !Ensembl reference genome, followed by differential gene expression for the sample groups.