Kapa hyper dna library prep kit

Genomic DNA and RNA were extracted from tumor FFPE samples using the QIAamp DNA FFPE Tissue Kit and the miRNeasy FFPE Kit, respectively. The fragment DNA was generated with Bioruptor (Diagenode, Bioruptor UCD-200). Ribosomal RNA was removed using RNase H followed by library preparation using the KAPA Stranded RNA-seq Kit with RiboErase (HMR) (KAPA Biosystems). Libraries were constructed using the KAPA Hyper DNA Library Prep Kit (KAPA Biosystem, KK8504). The dual-indexed sequencing libraries were PCR amplified with KAPA HiFi Hot start-ready Mix (KAPA, KK2602) for 4–6 cycles, then cleaned up by purification beads (Corning, AxyPrep Fragment Select-I kit, 14223162). Library concentration and quality were determined by the Qubit 3.0 system (Invitrogen) and Bioanalyzer 2100 (Agilent, Agilent HS DNA Reagent, 5067-4627), respectively.

For whole blood samples, plasma and leukocytes were separated from other blood cells by centrifuging (at 1900× g for 10 min at room temperature). Cell-free DNA was extracted from 2 mL plasma using QIAamp Circulating Nucleic Acid kit (Qiagen, Germantown, MD, USA). The construction of sequencing libraries was performed using the KAPA Hyper DNA Library Prep Kit (KAPA Biosystems, Wilmington, MA, USA). Dual-indexed sequencing libraries were amplified by polymerase chain reaction (4–7 cycles), followed by purification. The size of library fragment was determined by Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Customized probes targeting 139 cancer-relevant genes in lung cancer (Pulmocan^TM, Nanjing Geneseeq Technology Inc., Nanjing, China) were used for hybridization enrichment. Target-enriched libraries were then sequenced on Illumina sequencing platforms (Illumina, San Diego, CA, USA) as described previously [12 (link)]. The sequencing depths of the majority of plasma samples and leukocyte samples were at least 5000× and 200×, respectively.

Each embryo was transferred to lysate buffer in a PCR tube and immediately stored at − 80 °C. A diluted ERCC mix (Life Technologies 4456740) was added to the lysis buffer as a spike-in for each sample. Reverse transcription was performed directly on the cytoplasmic lysate. Terminal deoxynucleotidyl transferase was then used to add a poly(A) tail to the 3′-end of the first-strand cDNAs. The total cDNA library of the single cells was then amplified for 18–20 cycles to construct the library. The amplified cDNA library was fragmented using a Covaris sonicator (Covaris S220, Woburn, MA, USA). The KAPA Hyper DNA Library Prep Kit (KK8504, Kapa Biosystems) was used according to the manufacturer’s instructions to generate the sequencing libraries. Paired-end 150-bp sequencing was further performed on a HiSeq 2500 (Illumina) platform at the Annoroad Gene Technology Corporation, Ltd. (Beijing, China).

Tumor genomic DNA was extracted from whole blood and tumor biopsies, and fragmented DNA was generated with a Bioruptor (Diagenode, Bioruptor UCD-200) following the manufacturer's protocol. Libraries were constructed using the KAPA Hyper DNA Library Prep Kit (KAPA Biosystem, KK8504). Finally, dual-indexed sequencing libraries were PCR amplified with KAPA HiFi Hot start-ready Mix (KAPA, KK2602) for 4–6 cycles, and they were then cleaned up with purification Beads (Corning, AxyPrep Fragment Select-I kit, 14223162). The library concentration and quality were determined using the Qubit 3.0 system (Invitrogen) and Bioanalyzer 2100 (Agilent, Agilent HS DNA Reagent, 5067-4627).

The scRNA-seq method followed protocols established in previously published studies [52 (link)–54 (link)]. Briefly, the harvested single MEF, CC, MII oocyte, and pre-implantation embryos of the in vivo, NTM and NTC groups were washed several times in 0.5% BSA-PBS (Sigma) solution and subsequently selected and transferred into lysate buffer by a mouth pipette. A diluted ERCC mix (Life Technologies 4,456,740) was spiked into the lysis buffer of each sample, and reverse transcription was performed directly on the cytoplasmic lysate. Terminal deoxynucleotidyl transferase was then used to add a poly (A) tail to the 3′ end of the first-strand cDNA. The total cDNA library of the single cell was then amplified in 18–20 cycles for the library construction, and the amplified cDNA was fragmented using the Covaris sonicator (Covaris S220, Woburn, MA, USA). To generate the sequencing libraries, the KAPA Hyper DNA Library Prep Kit (KK8504, Kapa Biosystems) was used following the manufacturer’s instructions. All adapters are listed in Additional file 14. Paired-end 150-bp sequencing was further performed on the HiSeq 2500 platform (Illumina) at Annoroad Gene Technology Corporation., Ltd. (Beijing, China).

Library preparation for each sample was performed according to the manufacturer’s protocol. Briefly, ~ 1 μg DNA was randomly sheared into 150–200-base pair fragments using a Covaris M220 instrument (Woburn, MA, USA), followed by library construction with a KAPA Hyper DNA Library Prep Kit (KAPA Biosystems, Wilmington, MA, USA). The adaptor library was amplified and linked, and the total library was accurately quantified by Qubit DNA HS Assay Kit (Invitrogen, CA, USA). A library hybridization kit, SeqCap EZ MedExome Enrichment kits (Roche, Basel, CH), was used to capture target sequences and bead capture and elution hybridization libraries with Roche’s customized 1000 targeted gene probes (Roche, Basel, CH). To construct the targeted gene list, we referred to FoundationOne and Integrated Mutation Profiling of Actionable Cancer Targets (IMPACT), which were designed by two authoritative organizations, Foundation Medicine and Memorial Sloan Kettering Cancer Center (MSK), respectively, and all received FDA approval.
After amplifying the captured library by PCR, the constructed library was sequenced by an Illumina HiSeq Xten sequencer (San Diego, CA, USA). The average sequencing depth of tissue samples was 500 X. It could detect mutations with very low frequency to 0.1%.