title: RNA-Seq of iPS during differentiation to SMCs: 31.3 replicate, day 24 (RepSox)
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. Isolated total RNAs were quantified using Qubit ds DNA HS assay kit. PolyA+ mRNA libraries were prepared from 200ng-1g of total RNA using QIAseq Stranded RNA library preparation kit (Qiagen) according to manufacturers instruction. Libraries were sequenced on a NextSeq500 instrument (Illumina, San diego, CA, USA) using NextSeq 500/550 High Output Kit v2 (75 cycles). Reads were demultiplexed using bcl2fastq2 (v2.18.12) and adapters were trimmed using Cutadapt (v1.15).