Neon nucleofection system

CEM, CEMSS or their derivative cells (10⁵ cells) were infected with HIV-1_NL4-3 (50 ng of p24 antigen) with or without 1 μg ml⁻¹ Dox. Viral supernatants were collected periodically and p24 levels were measured as described above. In AZT experiments, H9 cells (10⁶ cells) were transfected with 200 pmol ASK1-targeted siRNA (HSS106458; Life Technologies) or control siRNA using Neon nucleofection system (Life Technologies) at 48 h before infection. Cells were then infected with 50 ng of AZT-resistant HIV-1_NLGRINFQ. After 2 days, cells were washed four times with PBS and then treated with 10 μM AZT (Sigma-Aldrich) for 24 h. The culture supernatants and cell lysates were then harvested and subjected to western blotting or infectivity analysis. Infectivity was calculated by measuring LTR-driven luciferase activity of TZM-bl indicator cells infected with normalized viruses.

Immortalized porcine fibroblasts [22 (link)] were cultured in DMEM (Gibco, Waltham, MA, USA) supplemented with 1% penicillin/streptomycin (Gibco), 10% fetal bovine serum (Gibco), 100 mM β-mercaptoethanol (Sigma, St Louis, MO, USA), and 1% nonessential amino acids (Gibco) at 37 °C with 5% CO₂. We transfected 3 × 10⁵ cells using the Neon Nucleofection system (Invitrogen, Carlsbad, CA, USA). Briefly, 500 ng of the PB-CMV-Target-AID-PERV(pol-gag) vector and 500 ng of the transposase vector (pCy43, provided by the Sanger Institute) were transfected at 1400 V for 20 ms, with a pulse number of 2. After 2 d of transfection, the fibroblasts were treated with 2 µg/mL neomycin (Sigma) for 10 d for antibiotic selection. After selection, single cells were subcultured in each well of 96-well plates and then expanded. Cell colonies from each single cell were analyzed by PCR to confirm the integration of the vector. The primer sequences are listed in Table 2.

For siRNA‐KD experiments, H9c2 cells were seeded at 10⁶ cells per 100 mm dish and transfected with 25 nM scrambled siRNA (Invitrogen AM4611), Rbfox2 siRNA (Invitrogen siRNA ID# s96620) or PTBP siRNAs (Qiagen cat# SI02649206 and SI04255146) using Lipofectamine RNAiMAX (Thermo Fisher Scientific). Cells were harvested 72 h post‐transfection for RNA or protein extraction. For rescue experiments, 3 × 10⁶ H9c2 cells were transfected with eGFP (Sigma‐Aldrich), human GFP‐RBFOX2 (transcript variant 3) (Addgene, plasmid #63086) or empty vector (pcDNA 5) together with scrambled or Rbfox2 specific siRNAs using Neon Nucleofection System (Thermo Fisher Scientific) as described previously.³⁸ (link) RNA was harvested 48 h post‐transfection.

We performed GUIDE-Seq as described previously.^{21 (link)} Briefly, HEK293T cells were electroporated in a 24-well plate with 500 ng of Cas9, 500 ng of sgRNA, 10 ng of mCherry plasmids, and 7.5 pmol of annealed GUIDE-Seq oligonucleotide using the Neon nucleofection system (Thermo Fisher Scientific). After 72 hours post-nucleofection, genomic DNA was extracted with a DNeasy Blood and Tissue kit (Qiagen 69504) according to the manufacturer’s protocol. DNA libraries were prepared using custom oligonucleotides described in Tsai, et al.^{21 (link)} Library preparations were done with original adaptors with each library barcoded for pooled sequencing. The barcoded, purified libraries were sequenced on a MiniSeq platform in a paired-end (150/150) run.
Raw sequencer output (BCL) was demultiplexed and aligned to hg38 using GS-Preprocess (github.com/umasstr/GS-Preprocess).^{43 (link)} This software also constructed a reference of UMIs unique to each read and merged technical replicate BAM files. Off-target analysis of this input was performed using the GUIDEseq Bioconductor package.^{44 (link)} Only sites that harbored a sequence with ≤10 mismatches relative to the gRNA were considered potential off-target sites. GUIDE-Seq read count data is indicated in Supplementary Figures 2–3.