Single-molecule sequencing of CRISPR-edited Xist

Cas9-cl36 were transduced with virus containing a scrambled sgRNA or 14 Xist-derived sgRNAs used for validation. For scrambled sgRNA control, RFP+ cells were FACS sorted without dox/puro selection. For 14 Xist-derived sgRNAs, RFP+ cells were FACS sorted 7 days after dox/puromycin selection. Genomic DNA from these cells were extracted. A ~6 Kb target region located at Xist 5′-end (Supplementary Fig. 1e and Supplementary Table 2) was amplified using barcoded primers and PrimeSTAR GXL DNA Polymerase (Takara Bio), purified with AMPure PB beads (Pacific Biosciences) and sequenced on a PacBio Sequel sequencing platform (Pacific Biosciences, RTL Genomics). Circular consensus (CCS) reads were obtained from standard Pacbio sequencing analysis pipeline using at least 3 subreads from the same circularized single DNA molecule. All CCS reads were aligned to Xist sequence using Blasr^{50 (link)} with 99.9% identity (minPctIdentity = 99.9) and the average mapping rate was 42%.