Nebnext

Pooled libraries for expression of dual sgRNAs were generated as detailed previously^{8 (link)} and cloned into pPapi (Addgene 96921). Briefly, 552 S. aureus oligonucleotides were mixed with 552 S. pyogenes oligonucleotides at 5 µM each for extension with NEBNext (New England Biolabs) with annealing at 48 °C. Purified dsDNA was purified and ligated using 100 cycles of Golden Gate assembly with 100 ng insert and 500 ng of vector using BsmBI and T7 ligase per reaction. Libraries were were isopropanol precipitated and transformed into STBL4 electrocompetent cells. The 300k library resulted in 304,704 total pairings whereby 286,225 of these perturbations were gene;gene KOs and 15,008 were gene;non-targeting KOs. At 24 h before transfection, a density of 18 × 10⁶ HEK293FT cells were seeded in each T175 flask in 30 ml of DMEM + 10% FBS. Transfection was performed using TransIT-LT1 (Mirus) transfection reagent. Beginning with 6 ml of Opti-MEM, a DNA mixture was prepared consisting of packaging plasmid pCMV_VSVG (Addgene 8454, 10 µg), psPAX2 (Addgene 12260, 40 µg), and the sgRNA-containing vector (e.g., pPapi, 40 µg). After a 20-min incubation the solution was added dropwise to the T175 flask and incubated for 6–8 h. Fresh media was added to the cells and collected 36 h later and snap frozen.

Experiments with NEBNext® (NEB), TruSeq® (Illumina), NEXFlex™ (Bioo Scientific), QIAseq (Qiagen), and SMARTer (Takara Bio) were performed following the manufacturers’ recommendations. For all kits, 1 pmol of the miRXplore™ Universal Reference (Miltenyi Biotec) was used as input to test accuracy in detection (Figs. 2 and 3). Brain total RNA (1 μg) was used for Fig. 3 for all kits with the exception of QIAseq, where 500 ng of total RNA was used as per the manufacturer’s recommendations. Libraries were prepared in triplicate for all experiments. To determine concentration and quality of libraries, all libraries were analyzed with an Agilent D1000 ScreenTape on a 2200 TapeStation instrument (Agilent) and then quantified with a Qubit dsDNA BR Assay kit on a Qubit 3.0 instrument.

Genomic DNA (2–5 μg) was subjected to mechanical fragmentation using a BioRuptor (Life Technologies, Carlsbad, CA, USA). The number of cycles was adjusted to obtain DNA fragments of a final average size of about 500 pb. Samples were then used to prepare sequencing-amenable TruSeq libraries (NEB-Next, New England Biolabs, Ipswich, MA, USA). Briefly, DNA fragments were made blunt-ended, phosphorylated, adenylated and Illumina-compatible adapters were ligated. After purification, barcoded sequences as well as Illumina-specific sequences were introduced by PCR, followed by quantitation of individual libraries. Libraries were then pooled and quantified again. A quality control of the pooled library made in bioanalyzer is shown in the figure, including an estimation of the percentage of non-overlapping reads that could be obtained using a 2x250 paired-end sequencing protocol. Library was qPCR-quantitated and brought to a final concentration of 10 nM. DNA was then denatured and equilibrated so that a final concentration of 18 pM of library was loaded onto a MiSeq v.3 flowcell (Illumina, San Diego, CA, USA) and sequenced using a 2x250 paired-end sequencing protocol to obtain more than 400x high quality coverage (1.9–2.5 Gb) with 84% of the bases showing a Q30 factor > 30. Reads were finally split according to barcodes and used for bioinformatics analysis.

Landoltia punctata clone 5635 (Lp5635, formerly DWC138) was received from the Rutgers Duckweed Stock Collective (RDSC; http://www.ruduckweed.org/). Lp5635 was collected, patted dry to remove excess water and flash frozen in liquid nitrogen. Frozen tissue was ground with a mortar and pestle in liquid nitrogen. High molecular weight (HMW) DNA was isolated using a modified Bomb protocol (Oberacker et al., 2019 (link)). DNA quality was assessed on a Bioanalyzer and HMW status was confirmed on an agarose gel. Libraries were prepared from HMW DNA using NEBnext (NEB, Beverly, MA, USA) and 2 × 150 bp paired end reads were generated on the Illumina NovaSeq (San Diego, CA, USA). Resulting raw sequence was only trimmed for adaptors, resulting > 60× coverage of the diploid L. punctata genome (350 Mb).

Total RNA was extracted from the cell culture pellet using TRIzol Reagent (Life technologies, UK) and ssRNA was removed by precipitation in 2 M lithium chloride (Sigma, UK) overnight as described (Maan et al., 2007 (link)). The dsRNA (8 μl) was denatured by heating at 95 °C for 5 min and the first cDNA strand was synthesised using SuperScript III RT (Life technologies, UK) and then the second strand was synthesised using NEBNext (New England BioLabs, UK) according to the manufacturers' instructions. Double stranded (ds) cDNA was purified using the Illustra GFX PCR DNA and Gel Band Purification kit (GE Healthcare, UK) and quantified with the Qubit dsDNA HS Assay kit (Life technologies, UK). The concentration of dscDNA was then adjusted to 0.2 ng/μl with 10 mM Tris-HCl, pH 8.0 buffer. Libraries were prepared using the Nextera XT library preparation kit and sequencing was performed using MiSeq Reagent kit v2 (Illumina, USA) on the MiSeq benchtop sequencer.

All tumors used for sequencing had a tumor cell content higher than 90% confirmed by neuropathological evaluation of the hematoxylin and eosin stainings. Human clinical samples and data were collected after receiving written informed consent in accordance with the Declaration of Helsinki and approval from the respective institutional review boards. Purified DNA was quantified using the Qubit Broad Range double-stranded DNA assay (Life Technologies, Carlsbad, CA, USA). Genomic DNA was sheared using an S2 Ultrasonicator (Covaris, Woburn, MA, USA). Whole-genome sequencing and library preparations were performed according to the manufacturer’s instructions (Illumina, San Diego, CA, USA or NEBNext, NEB). The quality of the libraries was assessed using a Bioanalyzer (Agilent, Stockport, UK). Sequencing was performed using the Illumina X Ten platform. Information on available sequencing data for all cases are summarized in Supplementary Data file 1.