Astrios cell sorter

For isolation of cell populations, tibialis anterior, quadriceps and gastrocnemius muscles were collected uninjured or 3 days after glycerol injection and digested with Dispase II (2.5 U/ml) (Roche), Collagenase B (0.2%) (Roche) and MgCl2 (5 mM) at 37 °C. Cells were then incubated at 4 °C for 30 min with antibodies against CD45 (Invitrogen, MCD4501 or MCD4528; dilution for both 1/25), CD31 (Invitrogen, RM5201 or RM5228; dilution for both 1/25), CD11b (Invitrogen, RM2801 or RM2828; dilution for both 1/25), CD34 (BD Biosciences, 560230 or 560238; dilution for both 1/60), Ly-6A–Ly-6E (Sca1) (BD Biosciences, 561021; dilution 1/150), α7-integrin (R&D, FAB3518N; dilution 1/30) and CD140a (eBioscience, 12–1401–81 or 17–1401–81; dilution for both 1/30). Antibody validation is provided on the manufacturer’s website. Specific cell subsets were isolated with a Beckman Coulter Astrios Cell sorter as described below and represented in supplementary figures S1 and S3A. MuSCs were identified as CD31⁻/CD11b⁻/CD45⁻/Sca1⁻/CD34⁺/Integrin α7⁺ and Fibro/Adipogenic progenitors (FAPs) were identified as CD31⁻/CD11b⁻/CD45⁻/Sca1⁺/CD34⁺/PDGFRα⁺. Lineage positive cells (Lin+) were identified as: CD31⁺/CD11b⁺/CD45⁺. CD31⁻/CD11b⁻/CD45⁻/Sca1⁺/CD34⁺/PDGFRα⁻ cells were also collected and named PDGFRα⁻ cells.

The sgRNA library was packaged into lentivirus as previously described (78 (link)). After packaging and titering the lentivirus, 2×10⁸ A549-CRISPR-SAM-IFN response reporter cells were seeded onto 15 cm plates (10 plates total). The next day they were transduced with the packaged sgRNA library at a multiplicity of infection (MOI) of 0.5. After 48 h, the transduced cells were split and half were collected as a transduction control, and the remaining half were plated back onto 15 cm plates. The next day, cells were treated with IFN-α2 (4×10³ U/mL) for 6 h. Cells were then collected and sorted on a Beckman Coulter Astrios cell sorter. Specifically, gates were set to sort sfGFP-negative cells as the population of interest, as well as sfGFP-positive cells as a control population of cells still capable of signaling. This screen was performed in duplicate. Genomic DNA was extracted from sorted cells using the Zymo Quick gDNA micro prep kit. PCR was subsequently performed using barcoded primers as previously described using the NEB Next High Fidelity 2x PCR master mix (78 (link)). PCR bands were gel purified using the Thermo GeneJET gel extraction kit. Samples were then sequenced by next-generation Illumina MiSeq using paired-end 150 bp reads.