GoKawiilIn utero electroporation
All animal procedures in this study were performed with approval from the St. Jude Institutional Animal Care and Use Committee. IUE was performed as described previously27, and plasmids were prepared with a NucleoBond Xtra Maxi Plus EF kit (Takara Biosciences). After anaesthesia with 4.5% isoflurane, pregnant CD1 mice at E16.5 were subjected to abdominal incision to expose the uterus. A DNA plasmid cocktail (1 μg μl−1 pBCAG-HA-ZRFUS1, 1 μg μl−1 pbCAG-eGFP-Luciferase, 1.5 μg μl−1 pX330-sgTp53, 2 μg μl−1 GLAST-PBase, 1.5 μg μl−1 mPlagl1 single-guide RNA, 0.5 μg μl−1 TrackerSeq library, FastGreen dye) was injected into the lateral ventricles with a glass pipette. Electric pulses were then delivered to the embryos by gently clasping their heads with forceps-shaped electrodes. Six 33-V pulses of 55 ms were applied at 100-ms intervals. The uterus was then repositioned into the abdominal cavity, the abdominal wall was sutured and the skin was stapled. Following birth, pups were monitored for clinical signs of tumour growth (such as seizures, circling, and head doming), as well being monitored by magnetic resonance imaging every 2 weeks. At end point, mice were collected for isolation of nuclei or immunofluorescence staining. In accordance with our St. Jude Institutional Animal Care and Use Committee protocol, end point was defined on the basis of a set of neurological symptoms (gait, hunching, kyphosis, squinting), and these limits were not exceeded in any of the experiments. Mice for isolation of nuclei were perfused with 10 ml cold PBS, and tumours were frozen in isopentane and stored at −80 °C. For survival curves, pups from a minimum of 2 mothers were included. Randomization and blinding were not applicable.
RGC isolation
RGCs were previously isolated from Ink4a-knockout mice with GFP expressed from the Blbp promoter using a Worthington Papain Dissociation system (LK003150). Cells were grown in neural basal medium (Invitrogen) supplemented with sodium pyruvate, glutamine, B27, N2, bFGF (10 ng ml−1) and rhEGF (20 ng ml−1) and tested for mycoplasma monthly. Cells were grown on treated cell culture dishes coated with Matrigel (Corning). RGCs were made ZR positive using a lentivirus generated by the Viral Vector Core at St. Jude. Mouse tumour cells were seeded into 10-cm plates 24 h before infection and infected with lentivirus with 8 μg ml−1 polybrene for 24 h. Infected cells were selected with 2 μg ml−1 puromycin for 3 days. ZR expression was confirmed by western blotting. Single-guide RNAs for Plagl1 were generated by the Center for Advanced Genome Engineering with an Addgene 52961 backbone, which included RFP. ZR-positive RGCs were infected by the same method and sorted for RFP using a BD FACSAria Fusion system. Knockout of more than 90% was confirmed by targeted deep sequencing.
OPC isolation
Primary OPC cultures were performed as previously described56,57. In brief, cortical tissues from E14.5 mouse embryos were collected, and neural stem cells were cultured as neurospheres for 4 days. Neural stem cells were dissociated and plated on poly-d-lysine-coated dishes at a density of 1.5 × 104 cells cm−2 in OPC media. They were subsequently infected with either ZR-Lenti-Cherry or control virus for 14 h. After viral infection, cells were maintained in OPC medium for 4 days before being collected for ATAC-seq and RNA-seq.
RNA-seq and ATAC-seq analyses
RNA-seq and ATAC-seq analyses were performed using Genialis Expression software (https://www.genialis.com) deployed locally on St Jude HPC infrastructure. Briefly, the RNA-seq pipeline run on the Genialis platform comprised the following steps. Raw reads were filtered to remove adaptors and poor-quality reads using BBDuk (v.37.9; https://sourceforge.net/projects/bbmap/). The resulting reads were mapped to the reference genomes (Ensembl 92) using STAR (v.2.7.0; RRID SCR_015899). FeatureCounts (v.1.6.3; RRID SCR_012919) was used for quantification of gene expression levels, followed by DEseq2 (RRID SCR_000154) for differential gene expression analysis. Genes with low expression (expression count summed over all samples of less than 10) were filtered out from the input matrix to DESeq2. The paired-end reads from ATAC-seq were trimmed using BBDuk (v.37.9) and mapped to reference genome mm10 using Bowtie2 (v.2.3.4.1). MACS2 (v.2.1.1.20160309) was then used to call peaks on the aligned reads using a P value cutoff of 0.01 (parameters –shift −75 –extsize 150 –nomodel –call-summits –nolambda –keep-dup all −P = 0.01).
TrackerSeq library generation and validation
TrackerSeq library cloning was carried out generally as described in ref. 19. In brief, the pCAG-SacB plasmid was digested with BstXI, and the 8-bit barcode was cloned into it using NEBuilder HiFi master mix (six reactions in total), followed by isopropanol purification. Purified reactions were electroporated into Endura DUOs (Lucigen) using a MicroPulser with program Ec1 (Bio-Rad). Four electroporations were carried out then recovered for 1 h at 37 °C in 2 ml of recovery media. Next, cells were plated overnight at 32 °C on 245-mm plates (Corning). The following morning, plates were scraped and cells were collected in Luria Broth (Miller), and library plasmids were purified using Endofree midiprep kits (Qiagen). For validation, 10 ng of the library plasmid prep was amplified using 2xPhusion (NEB) and sequenced by the Hartwell Center for Genome Sequencing Facility at St. Jude.
... continue reading