Magnetic beads

We previously described a new, simple method to isolate cardiac fibroblasts from the heart C57Bl/6 mice^{15 (link)}. Briefly, hearts were harvested after cold PBS perfusion through the left ventricle and kept on ice before further proceeding. Tissues were mechanically and enzymatically dissociated using magnetic beads (VWR) in 0.025 mg/mL of Liberase TM (Collagenase I and II, Roche) solution in pure Dulbecco’s modified Eagle’s medium (DMEM, Gibco) complemented with DNAse (40 μg/mL, Stemcell Technologies) on a magnetic shaker for 45 min at 37 °C. Cells were isolated by filtering the resulting cell suspension through a 70 µm cell strainer followed by a 500-rpm centrifugation to allow the deprivation of cardiomyocytes. Successively, supernatants were passed through a 40 µm cell strainer, centrifuged at 1400 rpm for 4′ and washed with PBS. Gp38⁺ cells were pre-selected with anti-gp38-APC staining (eBioscience) followed by magnetic anti-APC-microbeads (Miltenyi Biotec) staining and autoMACS® Pro (Miltenyi Biotec) separation. Finally, enriched gp38⁺ cells were further FACS-sorted or used for cell culture and in vitro assays.
Foetal human cardiac fibroblasts were purchased from Sigma (Cell Applications), and cells from passages 10–15 were used.

For Sanger sequencing of Arabidopsis amplicons, PCR was performed with Red Taq DNA Polymerase Master Mix (VWR Life Science) according to the manufacturer's instructions and purified with magnetic beads (CleanNGS). The PCR amplified regions were Sanger sequenced via Mix2Seq (Eurofins Genomics).
Sets of HiPlex amplicons were designed for Arabidopsis (40 amplicons), soybean (40 amplicons), maize (117 amplicons), and chicory (two assays with 45 and 49 amplicons, respectively). Genomic DNA for each species was submitted for HiPlex sequencing (Floodlight Genomics LLC). For Arabidopsis and soybean, the sequencing was done on 24 biological replicates of the reference genotypes. For maize, 20 wild-type leaf samples and 36 transfected protoplast samples (10 gRNA arrays × 3 replicates plus 6 Cas9-only negative controls) were sequenced. Six technical replicates of the genotype L9001 (C. intybus var. sativum) were used as controls for the pooled sequencing run and two technical replicates of the L9001 reference genotype were included in the individual plant sequencing run. Details of the pooling strategy can be found in Supplementary Table S4.