title: ATAC_Seq of iPS during differentiation to SMCs: 31.3 replicate, day 16 (RepSox), 2
design: At first day of differentiation (day 0), iPSCs were dissociated into single cells by Accutase (Thermo Fisher Scientific) for 8 mins at 37 oC. Cells were seeded on Matrigel-coated dish at 105 cells/cm2 and cultured in E8BAC medium (E5 medium plus 5 ng/ml BMP4, 25 ng/ml Activin A, 19.4 mg/l insulin, 10 M Y27632 and 1 M CHIR99021) for 36 hours (Day2). Then, cells were dissociated by Accutase and seeded on a new dish coating Matrigel at 1.6 x 104 cells/cm2 in E6T medium (E5 medium supplemented with 19.4 mg/l insulin, 1.7 ng/ml TGF-beta1 and 10 M Y27632) for 18 hours. Cells would achieve 100% confluence on day 6 for the optimal differentiation efficiency. From day 3 to day 7, cells were cultured with E5F medium (E5 medium supplemented with 19.4 mg/l insulin and 100 ng/ml FGF2). The E5F medium was changed at day 3, day 4 and day6. From day 8 to day 11, the cells were treated with FVR medium (E5 medium supplemented with 19.4 mg/l insulin, 50 ng/ml VEGF and 5 M RESV) and changed every other day. From day 12, cells started to differentiate into SMC phenotypes. For TP protocol, cells were cultured in E6-TP medium (E5 medium supplemented with 19.4 mg/l insulin, 5 M RESV, 10 ng/ml PDGFbb and 1.7 ng/ml TGF-beta1). For RepSox protocol, cells were treated with E6-R medium (E5 medium supplemented with 19.4 mg/l insulin, 5 M RESV, 25M Repsox). All media were changed every other day after day 12. On day 16, cells were cryopreserved when necessary or split in a new Matrigel-coated dish at 1x105 cells/cm2 cell density. The volume of medium was increased to 1.5x volume over standard volume for day 1 and day 8-24. After day 24, R-SMCs were maintained in E6R medium, TP-SMCs and primary SMCs were grown in DMEM supplemented with 5 g/ml insulin, 0.5 ng/ml EGF, 2 ng/ml FGF and 5% FBS. 60,000 cells were collected and washed in PBS. The transposition reaction was performed as described in the Omni-ATAC protocol. Then, the transposition reaction was purified with MinElute PCR Purification Kit (QIAGEN, Hilden, Germany). Tagmented fragments were isolated and PCR-amplified for 6-8 cycles as described previously7. Amplified DNA was purified using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA), according to manufacturers instructions. ATAC-seq libraries were sequenced using 42 paired-end sequencing cycles on an Illumina NextSeq500 system at the high throughput sequencing core facility of Institute for Integrative Biology of the Cell (CNRS, France, Centre de Recherche de Gif  http://www.i2bc.paris-saclay.fr/). Reads were demultiplexed using bcl2fastq2 (v2.18.12), and 43 to 175 million reads (fragments) were obtained per sample. Adapter sequences were trimmed using Cutadapt (v1.15).