Spri magnetic beads

Cortex homogenate was used for H3K27me3 ChIP. As for H3K9me3, Nuclei were extracted from the cortex of male offspring and neuronal (NeuN +) nuclei were enriched by FANS sorting using MoFlo Astrios EQ cell sorter (Beckman Coulter). Native ChIP was performed as described (Jiang et al. 2017 (link)). Briefly, the chromatin was digested with MNase at 28 ℃ for 10 min to obtain mononucleosomes and incubated with anti-H3K9me3 (Abcam AB8898) or anti-H3K27me3 (Millipore, 07–449) antibody at 4 ℃ overnight. The immunoprecipitated complexes were captured by protein A/G magnetic beads (Thermo Scientific, 88803) and washed with low-salt buffer, high-salt buffer, Lithium Chloride buffer and TE buffer. ChIP DNA was then eluted in elution buffer and incubated with RNase A, followed by proteinase K incubation. Finally, ChIP DNA was purified using SPRI magnetic beads (Beckman, B23318).
For ChIP DNA library preparation, End-repairing (Lucigen Corporation, ER0720) and A-tailing (Lucigen Corporation, KL11101K) was performed and then ChIP DNA was ligated (Lucigen Corporation, LK0750H) with Y-adaptor (Vazyme, N802) and subjected to PCR amplification (Vazyme, N618-01). Library DNA was size-selected with SPRI beads and sent to GENEWIZ,China, for deep sequencing with Novaseq set paired-end, 150 bp (PE150).

Viral RNA from neutralization-resistant viruses was then isolated using a QIAamp Viral RNA extraction kit (QIAGEN). The SNV M gene cDNA was amplified with a SuperScript IV One-Step RT-PCR kit (Thermo Fisher) using primers flanking the M gene. The resulting amplicon (~3,500 bp) was purified using SPRI magnetic beads (Beckman Coulter) and sequenced by the Sanger sequence technique using primers giving forward and reverse reads of the entire M segment. The full-length M gene segments for the original VSV/SNV stock used were also confirmed by Sanger sequencing. Each read was aligned to the VSV/SNV genome using Geneious Prime (v2020.1.2) to identify mutations.

Prior to sequencing, ChIP samples were library prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, NEBNext Ultra II DNA Library Prep Kit for Illumina, #E7770, NEBNext Multiplex Oligos for Illumina, #E7335L). Adaptor ligated DNA was size selected with SPRI magnetic beads (Beckman Coulter, B23318) which aims to retain DNA fragments between 200–300 base pairs (bp), recognizable for the Illumina sequencer (#NextSeq500). After library preparation, we performed qPCR, high sensitivity DNA quantification and size selection measurement (Agilent Bioanalyzer 2100 system + High sensitivity DNA measurement assay; 5067–4626) before sending samples for sequencing. Raw sequencing files processed by the Illumina NextSeq500 sequencer were obtained in “FASTQ” format. The raw sequencing files were then aligned to the genome using Bowtie 1.11 short reads sequence aligner using the human reference genome 19 (Hg19) as the reference genome. The output of Bowtie 1.1.1 is the “SAM” file extension format, for both input (control) and ChIP samples, which were then used by Model-based analysis for ChIPSeq (MACS) version 1.42 for peak calling; with all peaks called at a Q-value cut-off of 10⁻³ and default settings applied. MACS outputs result in “BED” file format and “WIG” files.