Lysis buffer

We conducted single-cell sequencing experiments on SD rats subjected to hSYN-GFP AAV2/9 and AAV2/9-hEF1a-GFP virus sciatic nerve injection in vivo and in vitro. The rats were euthanized by cervical dislocation, and the sciatic nerve was immediately immersed in ice-cold Dulbecco’s Phosphate-Buffered Saline (DPBS, Corning). The dissected sciatic nerve was cut from the distal end of the DRG to the end of the sciatic nerve (leaving out the DRG) and we carefully peeled away the epineural sheath in cold PBS. Collected sciatic nerves were cut into pieces under a fluorescence microscope. The GFP⁺ piece was collected and incubated in Hibernate A (BrainBits) containing papain (100 U; Sigma) at 37°C for 2 h with intermittent flicking. After removing enzymes, the collected pieces were trypsinized for 20 min at 37°C. The tissue was triturated into an individual cell suspension using a 1 ml pipette. We removed the trypsase and cellular debris with three rounds of mild centrifugation at 1000 × g and a Hibernate A minus Ca²⁺ and Mg²⁺ wash (BrainBits). The individual cell suspension was plated into a glass-bottom plate and collected using glass pipettes under a fluorescence microscope. The glass tip was broken off and left in each PCR tube containing lysis buffer (Vazyme Biotech) with water (2.4 μl), RNase-free DNase (0.2 μl), and murine origin RNase inhibitor (0.25 μl).

Total protein from tissue and cells were extracted in lysis buffer (Vazyme Biotech, Nanjing, China). Protein concentrations were determined using the Bio-Rad Protein Assay. Western blot analysis was performed as described [15 (link)]. Individual immunoblots were probed with primary antibodies, anti-DDX5 antibody (Cell Signaling Technology; Beverly, MA, USA; diluted 1:1000) and anti-β-Actin antibody (Santa Cruz Biotechnologies; Santa Cruz, CA, USA; diluted 1:3000).

Fluorescence-activated cell sorting machine (FACS Aria II, Becton Dickinson, New Jersey, USA) was used to sort a single cell into each well of a 96-well PCR plate containing 2.5μL of 10× Lysis Buffer (Vazyme# N711). For the isolation of macrophage, each pulmonary cell pool was stained with FITC-conjugated mouse anti-chicken MHC Class II and PE-conjugated mouse anti-chicken KUL01 monoclonal antibodies (Southern Biotech, Birmingham, USA). Macrophages were sorted following the manufacturer’s procedures. Herein, each sorted cell population was analyzed in four replicates, and 100 single cells were sorted in each replicate for subsequent SMART-Seq2 analysis. Four empty wells served as controls in each 96-well plate. Immediately after sorting, each plate was spun down to ensure immersion of cells into the lysis solution, snap-frozen on dry ice, and stored at −80°C until processing. Library construction and sequencing were completed by Gene Denovo (Guangzhou, China) as described previously [26 (link)]. Pearson’s correlation analysis was used to investigate the relationships between the SMART-Seq2 data of macrophages and lung clusters in scRNA-seq based on levels of gene expression.

Eight thousand sorted cells were harvested for genomic DNA extraction by addition of 20 μl of lysis buffer (Vazyme) following the manufacturer’s manual. For TIDER test, the genomic region in the vicinity of Cas nuclease target site was amplified by Phanta Max Super-Fidelity DNA Polymerase (Vazyme) using nested PCR. Purified PCR products were Sanger sequenced and analyzed as previously described^{44 (link)}. For deep sequencing analysis, the targeted genomic region was amplified by Phanta Max Super-Fidelity DNA Polymerase (Vazyme) using nested PCR, primers with barcode were used. PCR products were purified by Gel extraction kit (Vazyme) and sequenced on an Illumina HiSeq X System (150-bp paired-end reads). Forward reads were aligned to the reference sequences using BWA (v0.7.17-r1188) with parameter of “bwa mem -A2 -O3 -E1”. At each target, editing was calculated as the percentage of total reads containing desired edits without indels within a 10-bp window of the cut site. The target site informations are provided in Supplementary Table 2.

A fluorescence-activated cell sorting machine (FACS Aria II, Becton Dickinson, New Jersey, USA) was used to sort a single cell into each well of a 96-well PCR plate containing 2.5μL of 10× Lysis Buffer (Vazyme# N711). Pooled PBMCs were from the mixed equal amount of PBMC samples of three ALV-J infected chickens at 21 DPI. For the isolation of CD8^high+, CD8^medium+, and CD4⁺CD8^low+ (CD8^low+) populations, the pooled PBMCs from ALV-J infected chicken were stained for APC‐conjugated mouse anti‐chicken CD3⁺, FITC‐conjugated mouse anti‐chicken CD4⁺, and PE‐conjugated mouse anti‐chicken CD8α⁺ monoclonal antibodies (Southern Biotech, Birmingham, USA). For CD8^high αα⁺ and CD8^high αβ⁺ population isolation, the pooled PBMCs from ALV-J infected chicken were stained for APC‐conjugated mouse anti‐chicken CD3⁺, PE‐conjugated mouse anti‐chicken CD8α^+, and FITC‐conjugated mouse anti‐chicken CD8β⁺ monoclonal antibodies (Southern Biotech, Birmingham, USA) as described previously (Dai et al., 2020 (link)). Each population of five T lymphocyte subtypes was analyzed in four replications, and each replication sorted 100 single cells for subsequent SMART-Seq2 analysis. Furthermore, the gating strategy for each population was shown in Supplementary Figures 1A, B.