Hiseq novaseq platform

Tag-seq experiments were performed and analyzed as previously described [65 (link)]. Briefly, HEK293T cells were transfected by PEI with 20 nM Tag, 1000 ng of Cas nuclease, and 1000 ng single sgRNA or a pool sgRNAs (30–50 ng/sgRNA) per well in a six-well plate. A375 cells were transfected by Amaxa Cell Line Nucleofector Kit V (VCA-1003, LONZA, Switzerland) following the manufacturer’s instructions (2D) with 20 nM Tag, 1200 ng of Cas nuclease, and 800 ng WT/Mut-BRAF-sgRNA. All cells were harvested 3 days after transfection and genomic DNA was extracted for one-step libraries preparation by the Fragmentation, End Preparation, and dA-Tailing Module and Adapter Ligation Module kit (Vazyme Biotech Co., Ltd., Nanjing, China). The R and L libraries were constructed by PCR with library preparation primers, which were followed by sequencing (Hiseq/NovaSeq platform, Novogene, Beijing, China) and analysis with a Tag-seq bioinformatics pipeline. Tag-seq experiments were performed with the same input gDNA and an equal sequencing depth. The analysis pipeline is available at https://github.com/zhoujj2013/Tag-seq and 10.5281/zenodo.4679460.

Chromatin was extracted from cortices of P60 mice (two biological replicates per condition) injected with pAAV-Syn-DIO-Sa-dCas9-VPR and pAAV-U6-gRNA-Ctrl-Syn-DIO-EGFP (gRNA Ctrl condition; 1:2 rAAV ratio) or pAAV-Syn-DIO-Sa-dCas9-VPR, pAAV-U6-gRNA-4-Syn-DIO-EGFP, and pAAV-U6-gRNA-5-Syn-DIO-EGFP (gRNA 4+5 condition; 1:1:1 rAAV ratio) and prepared using the SimpleChIP Plus Enzymatic Chromatin IP Kit (cat. no. 9005, Cell Signaling) according to the manufacturer’s instructions. After chromatin shearing, immunoprecipitation was performed using an anti S. aureus Cas9 antibody (cat. no. C15200230, Diagenode). Library preparation and sequencing on the Illumina HiSeq/NovaSeq platform were performed by Novogene (Cambridge, UK). Sequencing reads were mapped to the genome using BWA.⁹⁰ (link) Mapping was restricted to reads that were uniquely assigned to the mouse genome (GRCm38.p6). Biological replicates were pooled to call peaks of gRNAs 4 + 5 versus gRNA Ctrl using MACS2.⁹¹ (link)

For deep transcriptome sequencing, 2-day-old mycelia of wild-type and ΔFgspe3 strain were harvested from liquid YEPD shaken at 180 rpm, washed three times, and ground in liquid nitrogen. For each strain, there are three biological replicates independently. Total RNA was isolated, and cDNA libraries were constructed by NEBNext^® Ultra^TM Directional RNA Library Prep Kit (New England Biolabs, Ipswich, MA, United States) using the manufacturer’s instructions. The cDNA libraries were sequenced by an Illumina Hiseq Novaseq platform with paired-end reads (150 bp) by Novogene Corporation (Beijing, China). Reads were mapped to the genome of F. graminearum PH-1 strain. DESeq R package was used for differential expression gene analysis by Novogene Corporation (Beijing, China). Genes with log₂ Fold change > 1 or < −1 and P-value < 0.05 were divided into differentially expressed. Raw data of the RNA-seq experiments generated in this article were deposited in the National Center for Biotechnology Information under the accession number PRJNA759842¹.