D1000 screentape kit

Briefly, genomic DNA was extracted from formalin-fixed, paraffin-embedded tumor tissues and whole blood according to the manufacturer’s standard protocol (Institute of Pathology, Southwest Hospital, Chongqing, People’s Republic of China). The concentration of the DNA samples was measured with the dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA) using a Qubit Fluorometer to ensure that genomic DNA was greater than 30 ng. Then, DNA shearing was performed using Covaris M220, followed by end repair, phosphorylation, adaptor ligation and polymerase chain reaction (PCR) amplification. The purified pre-enrichment library was hybridized to an OncoScreenPlus^TM (Burning Rock, Guangzhou, China) panel covering 520 human cancer-related genes as well as more than 9000 single-nucleotide polymorphisms (SNPs) located throughout the genome, followed by hybrid selection with magnetic beads, and PCR amplification. The quality and size distribution of the libraries were assessed by a dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA) using a Qubit Fluorometer and a High Sensitivity D1000 ScreenTape kit using 4200 TapeStation (Agilent Technologies, CA, USA). Indexed samples were then sequenced on a NextSeq sequencer (Illumina, San Diego, CA) with paired-end reads (read length, 150 bp) and an average sequencing depth of 1000× for tissue samples and 200× for whole blood samples.

Lymphocyte RNA was extracted using the trizol method. For quality control of RNA extraction yield and library synthesis products, RNA Screen Tape kit (Agilent Technologies, Waldbronn, Germany), D1000 ScreenTape kit (Agilent Technologies, Waldbronn, Germany), Qubit® RNA HS Assay kit (Invitrogen, USA), Qubit® DNA HS Assay kit (Invitrogen, USA) were used for each specific step. For mRNA library preparation, KAPA Stranded mRNA-Seq Kit with mRNA Capture Beads (Kapabiosystems, KK8421, https://www.kapabiosystems.com) was used. In brief, one μg was used for the library construction; the library was eluted in 20 μl of elution buffer. Libraries were adjusted to 10 Mm; 10 μl, 50% from each sample was collected and pooled in one tube. Multiplex samples Pool (1.5 pM including PhiX 1.5%) were loaded on NextSeq 500/550 High Output v2 kit (75 cycles) cartridge (Illumina, USA) and loaded on the NextSeq 500 System (Illumina, USA), with 75 cycles and single-Read Sequencing conditions.

As previously described⁴⁰ , for RNA extraction, frozen samples were homogenized in 1 ml TRIzol reagent (1000 μL per sample; Life Technologies, Grand Island, NY, USA) by using a tissue homogenizer (IKA labortechnik). DNA-free RNA was obtained by using an RNeasy Mini Kit (QIAGEN) with DNase treatment according to the manufacturer’s instructions. For quality control of RNA extraction yield, an RNA Screen Tape kit (Agilent Technologies), a D1000 Screen Tape kit (Agilent Technologies), Qubit RNA HS Assay kit (Invitrogen) and a Qubit DNA HS Assay kit (Invitrogen) were used for each specific step. All samples passed quality control analysis on a Bioanalyzer 2100 (Agilent Technologies). The 3 biological replicates with the highest RNA were used for mRNA library preparation and bioinformatics analysis.

Lymphocyte RNA was extracted using the trizol method. For quality control of RNA extraction yield and library synthesis products, an RNA Screen Tape kit (Agilent Technologies, Waldbronn, Germany), D1000 ScreenTape kit (Agilent Technologies, Waldbronn, Germany), Qubit^® RNA HS Assay kit (Invitrogen, USA) and Qubit^® DNA HS Assay kit (Invitrogen, USA) were used for each specific step. For mRNA library preparation, poly-A selection beads followed with NEXTflex™ Rapid Directional qRNA-Seq™ Kit were used (Bio Scientific, Cambridge, UK). In brief, 1 μg was used for the library construction and the library was eluted in 20 μL elution buffer. Libraries were adjusted and pooled to a concentration of 4 nM. Multiplex sample pools including PhiX 1.5% were loaded on a NextSeq 500/550 High Output v2 kit (75 cycles) cartridge (Illumina, San Diego, CA, USA) and loaded on the NextSeq 500 System (Illumina, USA), with 75 cycles and single-read sequencing condition.

RNA samples (n = 3) with an RNA Integrity Number above 8.0 were used for library preparation with the TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, USA) using 500 ng total RNA input. Library quality control was performed on the 4200 TapeStation with the D1000 ScreenTape kit (Agilent). cDNA libraries (n = 3) were sequenced on one lane of an Illumina NextSeq550 instrument utilizing paired-end 150-bp reads. Sequencing was performed at the Vienna BioCenter NGS Unit, Vienna, Austria.

Briefly, genomic DNA was extracted from formalin-fixed, paraffin-embedded tumor tissues and whole blood according to the manufacturer’s standard protocol (Institute of Pathology, Southwest Hospital, Chongqing, People’s Republic of China). The concentration of the DNA samples was measured with the dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA) using a Qubit Fluorometer to ensure that genomic DNA was greater than 30 ng. Then, DNA shearing was performed using Covaris M220, followed by end repair, phosphorylation, adaptor ligation and polymerase chain reaction (PCR) amplification. The purified pre-enrichment library was hybridized to an OncoScreenPlus^TM (Burning Rock, Guangzhou, China) panel covering 520 human cancer-related genes as well as more than 9000 single-nucleotide polymorphisms (SNPs) located throughout the genome, followed by hybrid selection with magnetic beads, and PCR amplification. The quality and size distribution of the libraries were assessed by a dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA) using a Qubit Fluorometer and a High Sensitivity D1000 ScreenTape kit using 4200 TapeStation (Agilent Technologies, CA, USA). Indexed samples were then sequenced on a NextSeq sequencer (Illumina, San Diego, CA) with paired-end reads (read length, 150 bp) and an average sequencing depth of 1000× for tissue samples and 200× for whole blood samples.