GSM8078724: HiC_CM_SRF_KO_Cre_rep2; Mus musculus; Hi-C
HiC was performed using a total of 5x105 cells per sample. Cells were treated and collected as follows. For non-differentiated ESCs, 2x106 Srf-flox1 cells encoding endogenous CTCF-FKBP were deprived of feeder cells and transfected with 25 μg GFP (Addgene, #11150) or 25 μg GFP-Cre plasmid (Addgene, #13776) using Lipofectamine 2000. 24 hours after the transfection, the medium was refreshed and supplemented with 250 nM dTAG13 or the matching volume of DMSO. After 24 hours of incubation with dTAG13 or DMSO, 5x105 of GFP-positive WT (GFP + DMSO), SRF KO (GFP-Cre + DMSO), CTCF KO (GFP + dTAG13), or SRF/CTCF dKO (GFP-Cre + dTAG13) cells were sorted using FACS Aria II. For dTAG13-induced SRF KO ESCs, 1x106 F1G4 cells encoding endogenous SRF-FKBP were treated with 250 nM dTAG13 for 24 hours, and 5x105 cells were collected using FACS Aria II. For CM cells, Srf-flex1 cells carrying H2B-mCherry (WT) or H2B-Venus-T2A-Cre (KO) were trypsinized at day 4 of cardiac mesoderm differentiation, and 5x105 cells expressing mCherry or Venus were sorted on a FACS Aria II. Sorted cells were crosslinked using Low Input crosslinking protocol provided by the Arima-HiC Kit user guide (A 160134 v01) and frozen in liquid nitrogen. Proximally-ligated DNA was generated using Arima-HiC Kit (A510008) following the manufacturer's protocol. HiC library was generated using KAPA Hyper Prep Kit (Roche, 07962312001) and KAPA single-indexed adapters (Roche, 08005702001) according to Arima's Library preparation user guide. Covaris S220 was used for DNA shearing. The libraries were quantified using the Qubit DNA HS assay and the library size was validated using DNA HS Bioanalyzer chips. Paired-end sequencing was performed on the HiSeq 4000 with 2x 75 bp read length or on the NovaSeq 6000 with 2x 100 bp read length.