Quick dna microprep plus kit

Human CRISPR Knockout Pooled Library (GeCKO V2, 1000000049) was purchased from Addgene. The libraries were amplified using Endura cells (catalog 60242, Lucigen). The workflow of CRISPR/Cas9 pooled screen is shown in Figure 1A. PLC cells were infected with lentiviral particles packaging Cas9 protein at an MOI less than 0.7. The blasticidin-selected Cas9-expressing PLC cells were infected with pooled lentiviral CRISPR library at an MOI of 0.3 (1,000× coverage) to ensure single-copy sgRNA integration in each cell. A pool of knockout cells was created after 7 days of selection with 2.5 μg/mL puromycin. CD24⁺CD133⁺ HCC CSCs and CD24^–CD133^– non-HCC CSCs were sorted by FACS. Genomic DNA of cells were extracted using Quick-DNA Microprep Plus Kit (catalog D4074, Zymo Research) according to the manufacturer’s instructions. Sequencing libraries were constructed and sequenced by GENEWIZ company. The sequencing data were analyzed by MAGeCKFlute (49 (link)).

DNA fractions were extracted using the Zymo QuickDNA Microprep plus kit according to the manufacturer’s instructions. Only samples with a total DNA yield higher than 10 ng were taken forwards for WGS library preparation. Libraries were prepared using the NEBNext Ultra II FS kit according to the manufacturer’s instructions. A short enzymatic fragmentation step of 5 min was performed, and five PCR cycles were used for library enrichment. After purification, libraries were quantified by Qubit and run on the Agilent Tapestation using HSD1000 screentapes. Samples with sufficient library DNA yield and characteristic fragment size distribution (about 200–500 bp) were further subjected to either low-pass (about 1× coverage) or deep (about 35× coverage) WGS.

Each sample was processed as a pool. Ten bees were placed in a 2mL microtube with 300 µL of DNA/RNA Shield (Zymo Research, Irvine, CA, USA) and crushed with a TissueLyser II (Qiagen, Hilden, Germany) for 3 min at 30 Hz, as previously reported [60 (link),61 (link)]. The obtained suspension was split into two aliquots, from which DNA and RNA were separately extracted. The extraction of the nucleic acids was performed with the Quick DNA Microprep Plus Kit (Zymo Research) and Quick RNA Microprep Plus Kit (Zymo Research). During the process, the modified manufacturer’s instructions for solid tissue processing were followed [19 (link),62 (link)]. The obtained nucleic acids were eluted in 100 µL of DNAase-RNase-free water and the extracts were stored at −80 °C until the qPCR assays.

Before extraction, all samples were washed with 95% ethanol to remove external microbial contaminations, and each sample was analysed individually. The sample was placed in a 2-ml microtube with 500 µl of DNA/RNA Shield (Zymo Research, Irvine, CA, USA) and crushed with a TissueLyser II (Qiagen, Hilden, Germany) for 3 min at 30 Hz, as previously reported (Cilia et al., 2019 (link); Nanetti et al., 2021b (link)). The obtained suspensions were split into two aliquots from which DNA and RNA were separately extracted.
The above-described procedures were accomplished by using a Quick DNA Microprep Plus Kit (Zymo Research) and Quick RNA Microprep Plus Kit (Zymo Research), respectively, following the modified manufacturer’s instructions for solid tissue processing (Mazzei et al., 2019 (link); Nanetti et al., 2021c (link)).
The obtained nucleic acids were eluted in 50 µl of DNAase-Rnase-free water, and the extracts were stored at −80°C until the qPCR assays.

The reference material used for this study was provided by the Czech Environmental Inspectorate and zoological gardens. The biological material came either from animals that died from natural causes in zoological gardens, or their excrements. The sampling did not involve the infliction of trauma to living animals. The research thus did not fall under the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes.
Human DNA samples were sampled and used in accordance with the Regulation (EU) 2016/679 of the European Parliament and of the Council of 27 April 2016 on the protection of natural persons with regard to the processing of personal data and on the free movement of such data, following Directive 95/46/EC (General Data Protection Regulation).
DNA extraction from the reference material (see Table 1) was performed using a Quick-DNA Microprep Plus Kit (ZymoResearch, Irvine, CA, USA) with final elution to 20 µL H₂O. The quantification of extracted DNA was achieved using UV–VIS or qPCR [18 (link)], where the nuclear DNA concentrations were measured by targeting the STR locus Pati01 [29 (link)].